Method of producing polyvalent antigens

ABSTRACT

Embodiments of the invention generally provide methods and compositions for producing polyvalent antigens. In one aspect, the invention provides a method for producing a cross-linked antigen. In another aspect, the invention provides a method of using cross-linked products as antigens to immunize animals and induce strong immune responses.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional patentapplication serial No. 60/361,166, entitled, “METHOD OF PRODUCINGNONTOXIC CROSS-LINKED ANTIGENS”, filed Mar. 1, 2002, and U.S.provisional patent application serial No. 60/363,445, entitled, “METHODAND USES OF PRODUCING POLYVALENT PEPTIDE ANTIGENS BY TRANSGLUTAMINASES”,filed Mar. 8, 2002. Each of the aforementioned related patentapplications is herein incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Vaccination has proven to be an effective way to prevent variousdiseases. Today, vaccines are available against many human pathogens,including bacteria, viruses, parasites, and fungi, and new vaccines areconstantly being developed.

[0003] Improvements to vaccine technology have focused on development ofvarious antigens to enhance immunogenicity and inducibility for bothcell-mediated immune responses and humoral immune responses. Forexample, for candidate antigens such as small protein subunits orpeptides having low immunogenicity, peptide aggregates and aggregates oflinked peptides have been tested as antigens for subunit vaccines.Linked peptide antigens currently being evaluated in clinical trialsinclude polymers of linked peptides from group A streptococcus as avaccine against rheumatic fever (Brandt et al 2000) and a plasmodiumpeptide polymer to induce a strong immune response against malaria(Nardin et al 2000).

[0004] Vaccination also can be used for treating noncommunicablediseases. For example, immunotherapy has been proposed to treatautoimmune diseases, viral infection-induced cancers, endogenous tumorantigens-induced cancers, and Alzheimer's Disease. The general conceptis to generate strong and persistent cytotoxic-T-cell responses againstvarious disease-associated antigens by immunizing an individual withdisease-associated antigens and/or antibodies against thedisease-associated antigens before (to prevent) or after (to treat)occurrence of the disease.

[0005] When developing vaccines, increasing the total amount of antigenhas proven to be problematic. Preparation of high potency antigens isneeded to minimize the total antigen content of the vaccines. It hasbeen shown that some antigens that have poor immunogenicity in monomerform were able to induce the production of antibody once they werecross-linked. For example, chemically cross-linked pertussis antigenshave induced higher immune response than monomeric pertussis antigens.(Watanabe et al, 2001). Despite this advantage, however, chemicalcross-linking typically involves a toxic cross-linking agent, such asglutaraldehyde, glyoxal, and octansdialdehyde.

[0006] Cross-linked protein crystals also have been proposed for use assafe and stable antigens. However, protein crystals are also prepared bychemical cross-linking and, further, have to be available in largequantities that require cumbersome manufacturing processes (Clair et al,1999).

[0007] Another drawback to improving vaccine technology throughcross-linking is that biological agents have limitations in that theyare very substrate specific; i.e., such agents react with only a limitednumber of compounds. Accordingly, biological agents have not been usedas cross-linking agents for preparing antigens or in other immunologicalapplications. Most known cross-linking biological agents such as enzymesor peptides are not desirable for use because of difficulties inproviding an adequate quantity, high cost, and difficulty inpurification of the agents. For example, cross-linking biological agentssuch as microbial transglutaminase has been purified mainly from culturemedium of the extra-cellular microbial transglutaminase. Suchpreparation is adequate for the appication of transglutaminases forcross-linking in industrial applications. However, microbialtransglutaminases purified from crude lysate or culture medium of batchfermentation may not be suitable for vaccine development and may includetoxic compounds and contaminating proteins which could induceundesirable cross-reactive antibodies.

[0008] Thus, although various biological agents such as enzymes havebeen employed in limited cross-linking applications, such as thepreparation of foods or cosmetics, methods for cross-linking a broadscope of proteins or peptides, particularly for therapeuticapplications, currently do not exist. Therefore, there exists a need formethods of producing biological agents which catalyze cross-linking on abroad scope of protein substrates. Such agents may be used, e.g., tocross-link proteins to be used as antigens for immunotherapy andantibody production.

SUMMARY OF THE INVENTION

[0009] The invention generally provides methods and compositions forproducing a polyvalent antigen using a biological agent. In oneembodiment, the method includes preparing an antigen in a cross-linkingsolution, combining the antigen cross-linking solution with a biologicalagent, and incubating the mixture at a temperature and for a period oftime sufficient to effect cross-linking of the antigen. The cross-linkedproducts are then used as a polyvalent antigen. In another embodiment,the antigen is actually more than one antigen, such as twotumor-associated antigens.

[0010] In yet another embodiment, the invention provides a method forproducing a polyvalent antigen using a transglutaminase as thebiological cross-linking agent. The method includes preparing an antigenin a cross-linking solution, combining the antigen cross-linkingsolution with the transglutaminase, and incubating the mixture at atemperature and for a period of time sufficient to effect cross-linkingof the antigen. Again, the cross-linked products may be used as apolyvalent antigen.

[0011] In yet another aspect of the invention, a method for producing anantibody against a compound is provided. First, the compound is preparedin a cross-linking solution. Then, the compound cross-linking solutionis combined with a biological agen. The mixture is treated at atemperature and for a period of time sufficient to effect cross-linkingof the compound into cross-linked products. The cross-linked productsare then administered to an animal under conditions that lead to anantibody response. The antibodies produced by the animal are thenisolated.

[0012] In an additional aspect of the invention, pharmaceuticalcompositions of the cross-linked products, antigen compositions, andantibodies of the present invention are provided. The invention alsoprovides a cross-linking solution for cross-linking a compound by anenzyme. The cross-linking solution includes at least one reducing agent,up to about 50% of glycerol, deionized water, and a pH-buffering agentfor adjusting the pH of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] So that the features of the invention can be understood indetail, a more particular description of the invention brieflysummarized above may be had by reference to embodiments illustrated inthe appended drawings. It is to be noted, however, that the appendeddrawings illustrate only certain embodiments of this invention shouldnot be considered limiting of its scope, for the invention may admit toother equally effective embodiments.

[0014]FIG. 1 is a simplified schematic of a method for cross-linkingcompounds using a biological agent.

[0015]FIG. 2 is a simplified schematic of a method for cross-linkingsynthetic compounds having lysine residues on one terminus and glutamineresidues on the other terminus using a transglutaminase.

[0016]FIG. 3 is a simplified schematic fo a method for producingtherapeutic agents using the methods of the present invention.

[0017]FIG. 4 is a polyacrylamide gel showing the results of purificationof recombinant SM TGase fusion protein.

[0018]FIG. 5 is a polyacrylamide gel showing the results of regenerationof enzymatic activity of the purified recombinant SM TGase.

[0019]FIG. 6 is a polyacrylamide gel showing the results ofcross-linking of β-amyloid peptide by purified recombinant TGase fusionprotein.

[0020]FIG. 7 is a polyacrylamide gel showing the results ofcross-linking of BSA5 peptide by purified recombinant TGase fusionprotein.

[0021]FIG. 8 is a graph of the ELISA results for anti-sera obtained fromusing cross-linked products of a protein mixture of serum albumin andcellulase as antigens and assayed against serum albumin.

[0022]FIG. 9 is a graph of the ELISA results for anti-sera obtained fromusing cross-linked products of a protein mixture of serum albumin andcellulase as antigens and assayed against cellulase.

[0023]FIG. 10 is a graph of the ELISA results for anti-sera obtainedfrom using cross-linked products of β-amyloid peptide as antigens.

[0024]FIG. 11 is a graph of the ELISA results for anti-sera obtainedfrom using cross-linked products of BSA5 peptide as antigens.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. All publicationsmentioned herein are incorporated by reference to disclose and describethe methods and/or materials in connection with the publications cited.In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

[0026] I. Definitions

[0027] The term “adjuvant” as used herein is defined as a substancecapable of non-specifically enhancing or potentiating an immuneresponse.

[0028] The term “antibody” as used herein is defined as animmunoglobulin protein made in response to a specific antigen. The term“antibody” encompasses all types of antibodies; e.g., monoclonalantibodies, polyclonal anti-sera, anti-serum having antibodies, etc.

[0029] The term “antigen” as used herein is defined as a moleculecapable of stimulating an immune response in an organism. An antigen ofthe invention includes but is not limited to an epitope, antigen, and/orantigenic fragment and could be a protein, a polypeptide, a peptide,etc. The term “antigenic fragment” as used herein is defined as aportion of a molecule capable of stimulating an immune response.

[0030] The term “antigenic determinant”, as used herein, refers to agiven region or three-dimensional structure of a molecule that bindsspecifically to an antibody.

[0031] The term “cross-linking” as used herein is defined as formationof a chemical bond within a molecule or between two molecules

[0032] The term “derivative”, as used herein, refers to a modified formof a compound.

[0033] The term “enzyme-linked immunosorbent assay” (ELISA) is a testthat detects antibodies based on a colorimetric reaction.

[0034] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of acompound or portions thereof and that mimics the chemical nature of thecompound.

[0035] The term “peptide” as used herein is defined as a short chain ofpolymerized amino acids or amino acid mimetics.

[0036] The term “protein” as used herein is defined as a polypeptidechain.

[0037] The term “purified antibody” as used herein is defined asantibody sufficiently free of the other proteins, carbohydrates, andlipids with which it is naturally associated.

[0038] The term “transglutaminase” as used herein is defined as anenzyme capable of catalyzing an acyl transfer reaction in which aγ-carboxyamide group of a peptide-bound glutamine residue is the acyldonor. The term “Ca²⁺-independent transglutaminase” as used herein isdefined as a transglutaminase active in the presence or absence of freeCa 2-ions; i.e., in the presence of excess ion chelators, such as EDTA.

[0039] II. Cross-Linking a Compound Using a Biological Agent

[0040] The invention relates to a method of cross-linking compoundsusing biological agents. FIG. 1 depicts a method 100 of cross-linking atleast one compound using a biological agent. At step 110, the compoundis prepared. Preparation of the compound includes, but is not limitedto, purification of native proteins, polypeptides, peptides or othercompounds to be cross-linked, biological synthesis or modification ofproteins, polypeptides or peptides by expression or over-expression ofbioengineered proteins, polypeptides or peptides, or chemicallymodifying or automatically synthesizing the compound to be cross linked.

[0041] Compounds that can be cross-linked by the methods describedherein include polypeptides, naturally occurring proteins, peptides,crude proteinaceous substances, or modified forms or mimetics of theaforementioned compounds with saccharides, fatty acids, steroids,purines, pyrimidines, structural analogs, derivatives, or combinationsthereof.

[0042] Other naturally occurring or synthetic molecule compounds thatcan be cross-linked and used in the methods herein include numerouschemical classes, though typically they are organic molecules, includingsmall organic molecules. Candidate compounds generally containfunctional groups necessary for a structural interaction with proteins,particularly hydrogen bonding; and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, and, preferably, at least two ofthese functional chemical groups. The candidate compounds may includecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups.

[0043] Typically, compounds to be cross-linked are substrates for thebiological cross-linking agents; however, in yet another aspect of theinvention, methods to alter, modify, or purify non-substrate compoundsare provided. One example of such a modification is through a specificpurification scheme. In another example of such a modification, theinvention provides a method of modifying compounds through the additionof functional groups or functional residues to the compounds. As aresult, many non-substrate compounds that are useful in a cross-linkedform can be cross-linked by the biological agents of the invention.

[0044] At step 120, the compound is cross-linked by a biological agent,for example, through an enzymatic reaction. As a result of thecross-linking activity of the biological agent, at step 130,cross-linked products of the compound are formed.

[0045] The cross-linking method 100 of the invention requiresspecificity, interaction, or recognition between the compound to becross-linked and the biological agent. For example, when a biologicalagent, such as an enzyme having cross-linking ability, is used, thecompounds to be cross-linked must be recognized by the biological agent.Generally, biological agents used for cross-linking include variousenzymes, purified from biological sources or synthesized de novo. Forexample, the biological agents may be obtained from any biologicalsource such as an animal, plant, or microorganism. The biological agentsmay be a naturally occurring enzyme, purified intracellularly orextracellularly in its native form, or may be a recombinant biologicalagent produced by using genetic engineering techniques or cellengineering techniques, or modified by protein engineering techniques orthe biological agent may be synthesized de novo.

[0046] Preferably, the biological agents chosen exhibit broad substratespecificity such that a broad range of compounds/substrates arecross-linked using the method 100. For example, microbialtransglutaminases, microbial lactases, and microbial bilirubin oxidasestypically have broadder substrate specificity than their counterparts inhigher organisms. Increasing the range of substrates that can becross-linked by a biological agent increases the value and usefulness ofthe agent.

[0047] Thus, in one aspect of the invention, the native or recombinantbiological agents used are altered, modified, or purified through aspecific purification scheme such that the modified biological agentsused have broader substrate specificity and cross-link a broad range ofcompounds, even compounds that are not natural substrates for thenon-modified biological agents.

[0048] The biological agents chosen may or may not require manyaccessory co-factors, coenzymes, or other factors, and preferably donot. For example, microbial transglutaminases do not require Ca²⁺, butother tansglutaminases may require Ca²+for cross-linking to occur.

[0049] Exemplary biological agents useful for the cross-linkingreactions of the present invention include but are not limited tovarious enzymes such as transferases, transglutaminases, oxidoreductases(i.e., enzymes classified under the Enzyme Classification number E.C. 1in accordance with the Recommendations (1992) of the International Unionof Biochemistry and Molecular Biology (IUBMB)), or combinations thereof.

[0050] An example of biological agents useful in methods of the presentinvention includes transglutaminases. Various types of transglutaminasesare known and vary depending on the source from which they are obtained.Suitable transglutaminases include but are not limited totransglutaminases derived from microorganisms (microbialtransglutaminase), fish transglutaminases, nematode transglutaminases,and mammalian transglutaminases. Microbial transglutaminases includetransglutaminases purified from microorganisms from the GenusStreptoverticillium, Bacilus, Steptomyces, etc., such as those reportedin Motoki et al, U.S. Pat. Nos. 5,156,956, titled “Transglutaminase”,filed on Jul. 1, 1991; and Washizu et al, Biosci. Biotech. Biochem.,58(1), 82-87 (1994), which are incorporated herein by reference.Exemplary mammalian transglutaminases include liver transglutaminase,plasma factor XIIIa, platelet placental factor XIIIa, hair-follicletransglutaminase, epidermal transglutaminase, cellular transglutaminase,tissue transglutaminase, nerve-derived transglutaminase, guinea pigliver transglutaminase, and prostate transglutaminase.

[0051] Other examples of biological agents include oxidoreductases (E.C.1), which are enzymes capable of catalysing redox reactions. Exemplaryoxidoreductases include laccases or related enzymes that act onmolecular oxygen (O₂) yielding water (H₂O) without peroxide (H₂O₂),oxidases or related enzymes that act on molecular oxygen (O₂) to yieldperoxide, and peroxidases or related enzymes that act on peroxide (e.g.H₂O₂) to yield water (H₂O).

[0052] Suitable oxidoreductases include but are not limited tosulfhydryl oxidases, lipoxygenases, phenolases, catechol oxidase (E.C.1.10.3.1), polyphenol oxidases (tyrosinase (E.C. 1.14.18.1)), laccases(Iysyl oxidases (E.C. 1.10.3.2)), bilirubin oxidases (E.C. 1.3.3.5),ascorbic acid oxidases (E.C. 1.10.3.3), ceruloplasmin (E.C. 1.16.3.1),peroxidase (E.C. 1.11.1), isomerases (e.g. proteindisulfide-isomerases), reductases (e.g. protein-disulfide reductases),and combinations thereof.

[0053] Thus, one embodiment of the invention provides methods forproducing biological agents, such as various enzymes, capable ofcross-linking a wide variety of compounds. In one aspect of thisembodiment, the biological agents used are purified intracellularly orextracellularly in their native form and used in this form or modified.The cross-linking biological agents may be purified through variousprotein purification procedures. Examples of purification proceduresinclude but are not limited to ammonia sulfate precipitation, salting inreactions, salting out reactions, and column chromatography employingthe principles of size-exclusion, cationic or anionic exchange, andvarious affinity interactions, etc. Alternatively, the biological agentsused are obtained by recombinant means. One embodiment of the inventionprovides methods of cloning and expressing the genes for the biologicalagents, and purifying recombinant forms of the biological agents usinggenetic engineering, cell engineering, or protein engineeringtechniques.

[0054] In one aspect of the invention, the biological agents used arekept in a reversibly inactive form and are activated into active formsduring cross-linking reaction. The invention provides a method ofproducing reversibly inactive forms of native or recombinant biologicalagents. The purpose of doing so is to avoid non-specific reactions orloss of activity during storage, to increase the expression level of thebiological agents, and to allow for the expression of the native orrecombinant biological agents without affecting the health and viabilityof the host cell. The reversibly inactive forms of the biological agentsare useful to target specific compounds to be cross-linked, and toobtain desirable cross-linked products.

[0055] The mechanism of the molecular weight-increasing, cross-linkingreaction by biological agents of the present invention is as follows. Ingeneral, functional groups of one or more compounds of the presentinvention are recognized and temporarily bound by the biological agentor agents to form intermolecular or intramolecular corss-linking bonds.For example, an oxidase catalyzes a reaction in which protons areremoved from a substrate in the presence of molecular oxygen, therebyforming oxidized products and water. As another example, atransglutaminase catalyzes a reaction in which an acyl group istransfered from an acyl donor compound to an acyl receptor on the sameor another compound. In the case of transglutaminases, intramolecular orintermolecular γ-glutamyl-ε-lysyl cross-linked products are formed.Typically, a γ-carboxy-amide group of a peptide-bound glutamine residueis the acyl donor and primary amino groups in a variety of compoundssuch as peptides, proteins, nucleic acids and similar compounds, such asthe ε-amino group of a lysine residue in a peptide or polypeptide chain,may function as acyl acceptors.

[0056] Typical functional groups that can be oxidized oracyl-transferred are found in the amino acid side chains of proteins,peptides, nucleic acids, and similar compounds. Exemplary functionalgroups include, but are not limited to, amines, carbonyl, hydroxyl orcarboxyl groups, including the γ-carboxy-amide group of a glutamineresidue, the ε-amino group of a lysine residue, the hydroxyl group oftyrosine, the sulfhydryl group of cysteine, and the imidazole group of ahistidine residue, as well as primary amino groups in a variety ofcompounds, such as peptides, proteins, nucleic acids, and similarcompounds, and combinations thereof.

[0057] The reaction of the compound with the biological agent (FIG. 1,step 120), may take place in the form of a solution, slurry or paste,but the reaction conditions and concentrations of both the biologicalcross-linking agents and the compounds to be cross-linked are selecteddepending on the properties of the reactants and cross-linked productsof interest. For example, the amount of the biological agents andcompounds to be used, the time and temperature of the reaction and thepH of the reaction solution are varied as necessary. In addition, such asolution, slurry or paste of the reactants may be obtained not only inaqueous form but also as an emulsion with an oil or fat and, asnecessary, or may be blended with additives such as salts, saccharides,proteins, perfumes, moisture keeping agents, and coloring agents.

[0058] III. Production of Biological Agents and Compounds

[0059] The invention provides recombinant biological cross-linkingagents and methods for producing and purifying recombinant biologicalcross-linking agents in vitro through recombinant DNA technology.Furthermore, the invention also provides recombinant compounds to becross-linked, and methods of producing and purifying the compounds to becross-linked by the biological agents, either in native form or usingrecombinant means.

[0060] Host cells transformed with nucleic acid sequences encoding thebiological agents or compounds of the invention may be cultured underconditions suitable for expression and recovery of the biological agentsor compounds from cell cultures. The recombinant biological agents andcompounds of the invention produced may be secreted or containedintracellularly depending on the nature of the biological agent orcompound and/or the vector used. They may be expressed as soluablecompounds or agents, or as insoluble aggregates or inclusion bodies. Forexample, expression vectors containing polynucleotides that encode thebiological agents and compounds of the invention may be designed tocontain signal sequences which help to direct secretion of thebiological agents and compounds through a prokaryotic or eukaryotic cellmembrane and into extracellular environments or culture media. Asanother example, a host cell line may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed proteins or peptides in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the protein may also be used to facilitate correct insertion,folding and/or function. Different host cells such as CHO, HeLa, MDCK,HEK293, and W138, which have specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

[0061] In addition, recombinant constructions known in the art may beused to join all or portions of the nucleotide coding sequences for thebiological agents or compounds to be cross-linked to nucleotidesequences encoding other polypeptide domains. The polypeptide domainscan be used to facilitate the purification of the biological agents andcompounds of the invention. Such purification-facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). In addition, it maybe useful to include cleavable linker sequences between the codingsequences and the purification facilitating domains, such as thosespecific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.)to facilitate purification and separate the purification-facilitatingdomains after purification.

[0062] For example, using the purification methods described herein, theinvention provides recombinant transglutaminases (TGase) fromStreptoverticillium mobaraense (ATCC 29032) and Streptoverticilliumcinnamoneum (ATCC 11874) that are overexpressed in E. coli and have beenpurified in vitro with a better yield, higher purity, and higherenzymatic activity than has been possible previously. Until now,identifying a safe, efficient, and cost-effective method of producingrecombinant TGase has met with little success. Native TGase purifiedfrom natural sources, such as the secreted bacterial TGase from culturemedium, has two primary disadvantages. First, contamination fromimpurities and pathogens is a problem. Yet, current methods of producingrecombinant microbial TGase using various expression vectors and/orchemically-synthesizing the coding sequences according to the preferredcodon usage of the host E. coli cell generally results in low enzymaticactivity, low yield, high cost, and protein precipitate/aggregateformation during purification (Washizu et al., Biosci Biotechnol Biochem1994, Takehana et al., Biosci Biotechnol Biochem 1994, EP 0,481,504 andU.S. Pat. Nos. 5,420,025 and 6,013,498). Secretion expression of TGaseby E. coli, yeast or the like, results in a yield that isdisadvantageously very small despite the use of large scale cellcultures, such as large fermentation equipment. Further, it has beenfound that since bacterial TGase is independent on calcium, theexpression of active TGase is fatal to the microorganism because theenzyme acts on proteins necessary for the survival of the host cells.

[0063] For the reasons above and for other reasons, the inventionprovides improved, novel methods of producing recombinant biologicalagents and recombinant compounds. For example, recombinant TGase,recombinant serum albumin, recombinant cellulase, recombinant bovineserum albumin (BSA), recombinant tumour necrosis factors (TNF-α), andrecombinant epidermal growth factor receptor (EGF-R), are among theuseful biological agents and compounds prepared using the purificationmethods of the invention. The methods are efficient, cost-effective tobe used among various biological agents and compounds to be cross-linkedas described herein, and the products are safe for pharmaceutical ormedical uses.

[0064] Isolation of genomic DNA of the biological agents and compoundsto be cross-linked can be accomplished by methods known in the art.Conventional methods and commercial kits are readily available to purifygenomic DNA. Alternatively, genes for the biological agents andcompounds to be cross-linked may be obtained as a cDNA, by cloning andscreening methods known in the art, for example, by constructing andscreening various DNA libraries, direct PCR cloning, and otherrecombinant means.

[0065] As mentioned previously, methods well known to those skilled inthe art may be used to construct cloning vectors containing appropriatetranscriptional and translational control elements and DNA sequences.Exemplary techniques are described in Sambrook, J. et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y., and Green, E. etal. (1997) Genome Analysis, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y.

[0066] In order to carry out certain aspects of the invention, primersmay be used to amplify the genomic or cDNA sequences of the biologicalagents and compounds to be cross-linked. For example, DNA fragmentscontaining all or portions of the transglutaminase coding sequences maybe used as probes for cloning of other transglutaminase (TGase) genes.For instance, SEQ ID No. 1 and SEQ ID No. 2 are provided herein asprimers for cloning of transglutaminase genes from Streptomycesmobaraensis ATCC 29032 (SM TGase). Also provided are SEQ ID No. 3 andSEQ ID No. 4 for cloning of transglutaminase genes from Streptomycescinnamoneus ATCC 11874 (SC TGase). Also provided are SEQ ID No. 5 andSEQ ID No. 7, which are the DNA sequences encoding the mature TGaseproteins from Streptomyces mobaraensis ATCC 29032 and Streptomycescinnamoneus ATCC 11874, respectively. In addition, SEQ ID No. 13 and SEQID No. 14 are provided as primer pair for cloning of cellulase gene fromHumicola grisea var. thermoides ATCC 16453.

[0067] A genomic sequence of interest may include nucleic acid sequencespresent between the initiation codon and the stop codon, containing allof the introns that are normally present in a native chromosome. It mayfurther include the 3′ and 5′ untranslated regions found in the maturemRNA. It may further include specific transcriptional and translationalregulatory sequences, such as promoters, enhancers, etc., includingabout 1 kb to 10 kb or more of flanking genomic DNA at either the 5′ or3′ end of the transcribed region. Genomic DNA may be isolated as a DNAfragment of 100 kb or smaller that is substantially free of flankingchromosomal sequence. Sequences required for proper tissue and stagespecific expression also can be cloned from genomic DNA flanking thecoding region (either 3′ or 5′) and/or internal regulatory sequences,sometimes found in introns.

[0068] The sequence of the 5′ flanking region may be modified to effectpromoter elements and/or enhancer binding sites, to providedevelopmental regulation in tissues where the gene of interest isexpressed. Tissue-specific expression is useful for determining thepattern of expression of the gene, and for providing promoters thatmimic the native pattern of expression. Naturally-occurringpolymorphisms in the promoter region are useful for determining naturalvariations in expression, particularly those that may be associated withdiseases.

[0069] Alternatively, mutations may be introduced into the promoterregion to alter the expression of the nucleic acid sequence. Methods forthe identification of specific DNA motifs involved in the binding oftranscriptional factors are known in the art, e.g., sequence similarityto known binding motifs, gel retardation studies, etc. For examples, seeBlackwell et al. (1995) Mol Med 1: 194-205; Mortlock et al. (1996)Genome Res. 6: 327-33; and Joulin and Richard-Foy (1995) Eur J. Biochem232: 620-626. Regulatory sequences may be used to identify cis actingsequences required for transcriptional or translational regulation ofthe expression of the biological agents and compounds of the invention,especially in different tissues or stages of development, and toidentify cis acting sequences and trans acting factors that regulate ormediate gene expression. Such transcription or translational controlregions may be operably linked to a gene for the biological agents andcompounds in order to promote expression of wild type or altered genesof interest in cultured cells, or in embryonic, fetal, or adult tissues,and for gene therapy.

[0070] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site-specific mutagenesis may be found inGustin et al. (1993) Biotechniques 14:22; Barany (1985) Gene 37:111-23;Colicelli et al. (1985) Mol Gen Genet 199:537; and Prentki et al. (1984)Gene 29:303-13. Methods for site specific mutagenesis can be found inSambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 15.3-15.108; Weiner et al., Gene 126:35-41 (1993); Sayers et al.Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990);Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu, Anal Biochem177:120-4 (1989).

[0071] The nucleic acid compositions of the invention may encode all ora part of the polypeptides for the biological agents and compounds to becross-linked. Double- or single-stranded fragments of the DNA sequencemay be obtained by chemically synthesizing oligonucleotides inaccordance with conventional methods, by restriction enzyme digestion,by PCR amplification, etc. For the most part, DNA fragments will be ofat least 15 nucleotides, usually at least 18 nucleotides or 25nucleotides, and may be at least about 50 nucleotides. Small DNAfragments are useful as primers for PCR, hybridization screening probes,etc. Larger DNA fragments, i.e., greater than 100 nt, are useful forproduction of a protein or polypeptide.

[0072] Altered nucleic acid sequences encoding the biological agents andcompounds to be cross-linked may include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functional equivalent of the compounds to becross-linked. The encoded protein may also contain deletions,insertions, or substitutions of amino acid residues, which producesilent changes and result in functionally equivalent compounds that canbe be cross-linked. The altered nucleic acid sequences for thebiological agents and compounds of the invention may be used to generatechanges in promoter strength or sequences of the encoded proteins, forexample, to promote folding of the encoding proteins, or to decreasesubstrate fidelity. Deliberate amino acid substitutions may be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of compounds to be cross-linked is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine. Such alterations to the compoundto be cross-linked may be made to increase expression, allow forpurification, or to add cross-linking groups to make the compound morereactive to the biological cross-linking agent.

[0073] In order to obtain biological agents in compounds to becross-linked, cloning of the genes encoded for biological agents andcompounds of the invention into an expression vector may be necessary.An expression vector may contain necessary elements for transcriptionand/or translation of the inserted coding sequences. Expression vectorsand systems known in the art may be employed for producing full lengthor only portions of the polypeptides of the biological agents andcompounds of the invention.

[0074] For long-term, high-yield production of recombinant proteins,stable expression of the DNA construct of biological agents and/orcompounds to be cross-linked is preferred. For example, cell lines whichstably express the biological agent and/or compounds may be transformedusing expression vectors which may contain viral origins of replicationand/or endogenous expression elements and a selectable marker gene onthe same or on a separate vector. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells, which successfully express theintroduced sequences. Resistant clones of stably transformed cells maybe proliferated using tissue culture techniques appropriate to the celltype. As another example, a host cell strain may be chosen for itsability to modulate the expression of the inserted sequences or toprocess the expressed proteins or peptides in the desired fashion. Suchmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the protein may also be used to facilitate correct insertion,folding and/or function. Different host cells such as CHO, HeLa, MDCK,HEK293, and W138, which have specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

[0075] With the availability of the protein or fragments in largeamounts, the recombinant biological agents and compounds to becross-linked may be isolated and purified in accordance withconventional methods. Again, see Sambrook, J., et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & sons, New York, N.Y. A lysate may be prepared of theexpression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification techniques. The purified proteins will generally be atleast about 80% pure, preferably at least about 90% pure, and may be upto and including 100% pure. Pure is intended to mean free of otherproteins, as well as cellular debris.

[0076] Production of the recombinant biological agents and compounds tobe cross-linked may be as insoluble inclusion body fusion proteins. Forexample, expression of recombinant transglutaminase proteins may betoxic to a host cell; thus an expression vector for highlevel-expression of insoluble protein is chosen to avoid the expressionof soluble active transgluamineases. Alternatively, genomic DNA encodingthe mature proteins for the biological agents and compounds to becross-linked are produced and isolated without signal peptides in orderto express the recombinant proteins inside the host cells withoutprocessing through the secretory pathway of the host cells. For example,when purifying mature recombinant TGase proteins having cross-linkingactivity, it is found that the expression of secreted TGases may betoxic to the host cells, reactive to the host proteins, and/or selfreactive, resulting in low yield and low activity of recombinant TGaseproteins.

[0077] In yet another approach, natural, modified, or recombinantnucleic acid sequences encoding the biological agents and the compoundsto be cross-linked may be ligated to a heterologous sequence to encode afusion protein. For example, it may be useful to encode chimericproteins that can be recognized by commercially available antibodies. Afusion protein may also be engineered to contain a cleavage site locatedbetween the encoding sequences for the biological agent and thecompounds, and the heterologous protein sequences, so that thebiological agent and the compounds may be cleaved and purified away fromthe heterologous moiety.

[0078] In summary, nucleotide sequences of biological agents and/orcompounds to be cross-linked can be engineered using methods generallyknown in the art in order to alter coding sequences for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing, and/or expression of the gene product, and,specifically, to decrease substrate specificity of the biological agentsand/or add cross-linking sites to the compounds to be cross-linked.

[0079] IV. Purification, Inactivation, Storage and Reactivation ofBiological Agents and Compounds to be Cross-linked

[0080] One embodiment of the invention provides cloning and purificationof recombinant biological agents and compounds to be cross-linked. Forexample, in bacterial systems, a number of expression vectors thatdirect expression of fusion proteins such that they are easily purifiedmay be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding the biologicalagent and compounds of the invention may be ligated into the vector inframe with sequences for the amino-terminal Met and the subsequent 7residues of β-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.264:5503-5509); and the like. pGEX vectors (Promega, Madison, Wis.) mayalso be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

[0081] As an example, for ease in purification, expression ofrecombinant transglutaminases can be done by cloning of genomic TGasegene into an inducible pET expression vector, combined with a6×-histidine-tagged fusion protein system. Such an expression vectorprovides for expression of a fusion protein containing the codingsequence of a biological agent or candidate compound of the inventionfused to a nucleic acid encoding six histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying the recombinant transglutaminase. A discussion ofexpression vectors for constructing fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453). Otherpurification techniques include but are not limited to ligand affinitychromatography, antibody affinity chromatography, ion-exchangechromatography, hydrophobic interaction chromatography, ultrafiltrationreverse phase high-performance liquid chromatography, isoelectric pointelectrophoresis, etc. Exemplary techniques are described in Scopes, R.K. (1994) Protein Purification: Principles and Practice (Third Edition),Springer-Verlag, New York, N.Y.

[0082] Thus, one aspect of the invention provides recombinant6×-histidine-tagged TGase fusion proteins, including SEQ ID No. 9, a DNAsequence encoding a recombinant 6×-His-TGase fusion protein ofStreptomyces mobaraensis ATCC 29032, and SEQ ID No. 11, a DNA sequenceencoding a recombinant 6×-His-TGase fusion protein from Streptomycescinnamoneus ATCC 11874. The invention also provides SEQ ID No. 10, atranslated amino acid sequence encoding the recombinant 6×-His-TGasefusion protein of Streptomyces mobaraensis ATCC 29032 (about 355 aminoacids), and SEQ ID No. 12, a translated amino acid sequence encoding therecombinant 6×-His-TGase fusion protein of Streptomyces cinnamoneus ATCC11874 (about 355 amino acids).

[0083] As another example, recombinant serum albumin can be expressedfrom an expression vector contiaining a cloned serum albumin insert assecreted recombinant proteins and purified from the extracellularcultures medium through ammonia sulfate percipitation. Another exampleof purification of compounds to be cross-linked through recombinant meanis the purification of recombinant cellulase. Genomic DNA of a cellulasegene was subcloned into pET expression system (Novagen) forover-expression as intracellular recombinant protein inclusion bodiesand purification through fusion protein purification techniques. Theinvention provides SEQ ID No. 13 and SEQ ID No. 14 as primers forcloning of cellulase gene.

[0084] In another embodiment of the invention, recombinant cross-linkingbiological agents and compounds to be cross-linked are purified in aninactive form to prevent cross-linking reaction during storage. Thus,the invention provides inactive forms of recombinant biological agentsthat can be reversibly reactivated into active forms. Such a treatmentis advantageous as it prevents self-cross-linking activity of thebiological agent during purification and storage, which often results inlow purification yield, loss of enzymatic activity, and precipitation ofprotein aggregates. Conditions have been optimized for inactivating andreactivating recombinant TGases; however such purification methods canbe applied to other biological agents and/or compounds to becross-linked.

[0085] The method involves first purifying the recombinant biologicalagents or compounds to be cross-linked under denaturing conditions usinga denaturant, such as solubilizing a protein inclusion body produced asdescribed above with a high concentration of guanidine, such as about 6Mto about 9M of guanidine, preferably about 8M of guanidine, prepared ina pH buffering agent, e.g., tris buffer titrated with hydrochloric acidto a pH of about 6 to about 8. In addition to a denaturant, the solutionfor solubilizing includsion bodies may also contain other salts, such asup to 0.8M of sodium chloride (NaCI) or potassium chloride (KCI), e.g.,about 0.2M of NaCl or about 0.5M of NaCl. One example of such lysis orsolubilizing buffer includes about 6M guanidine titrated withhydrochloric acid (guanidine-HCl) to a pH of about 7.9, prepared in asolution containing about 20 mM of Tris buffer, about 5 mM imidazole,and about 0.5M sodium chloride (NaCl). Other denaturants, such as about6 M to about 8 M of urea may also be used.

[0086] In one embodiment, the purification of the recombinant biologicalagents or compounds to be cross-linked is done in one single column stepusing affinity column chromatograpgy. After binding to an affinitycolumn, washing is generally performed under less stringent conditions,i.e. low salt or low ionic strength conditions. One example of a wahsingbuffer includes about 6M guanidine titrated with hydrochloric acid(guanidine-HCl) to a pH of about 7.9, prepared in a solution containingabout 20 mM of Tris buffer, about 20 mM imidazole, and about 0.5M sodiumchloride (NaCl). After washing, elution of the purifed recombinantproteins is generally performed under high stringent conditions, i.e.high salt or high ionic strength conditions. One example of a elutionbuffer includes about 6M guanidine titrated with hydrochloric acid(guanidine-HCl) to a pH of about 7.9, prepared in a solution containingabout 20 mM of Tris buffer, about 0.5 M imidazole, and about 0.5M sodiumchloride (NaCl).

[0087] After the recombinant biological agents or compounds to becross-linked were purified in the presence of a denaturant therebydeactivating them, they are refolded, preferably without regeneration ofactivity. Removing the denaturant and refolding the recombinantbiological agents or compounds to be cross-linked is accomplished bydiluting the volume of the purified recombinant proteins throughdialysis, ultrafiltration, or the like, preferably in a solution otherthan an activation solution to keep them in inactive form. Suitabledilution or dialysis solutions include, but are not limited to,phosphate buffered saline (PBS), Tris-HCl buffer with low concentrationsof salts (e.g., sodium clloride, potassium chloride, and others), etc.After dialysis, an additional concentrating step may be required toconcentrate the recombinant protein solutions; for example, usingcommercially available concentrators or dialysing in a storage bufferhaving high concentration of glycerol (e.g., about 20% or higher, suchas about 50% or higher). The purified recombinant proteins can bepurified to at least about 95% homogeneity, as judged by Coomassie Bluestaining and silver staining of a SDS-PAGE gel.

[0088] In one embodiment of the invention, the refolding, dilution ordialysis solution used does not include a reducing agent, including, butnot limited to, DTT, glutathione, etc., at a concentration up to about0.5 M. One example of a refolding solution includes about 0.75 M ofarginine, about 50 mM of Tris base titrated with hydrochloric acid(Tris-HCl) to a pH of about 8.0, about 50 mM of potassium chloride(KCl), and about 0.1 mM of a metal chelator, such as EDTA. Anotherexample of a refolding solution includes about 100 solution volumes ofPBS solution. One example of a dialysis solution includes about 50 mM ofTris-HCl (at a pH of about 7.9), about 50 mM of KCl, about 0.1 mM ofEDTA, and about 50% of glycerol. These steps or reactions can be kept toincubate at low temperature, such as at around room temperature or less,such as about 4° C., for more than one hour, such as about 48 hours.

[0089] The invention also provides storage buffer for the inactivebiological agents. For example, the storage buffer includes up to about200 mM of a salt, up to about 5 mM of a metal chelator, up to about 70%glycerol, and up to about 200 mM of Tris base titrated with hydrochloricacid, acetic acid, or other titration acid/base, to a pH of about 5 toabout 11. In another embodiment, the storage buffer used does notinclude a reducing agent, including, but not limited to, DTT,glutathione, etc., at a concentration up to about 0.5 M.

[0090] As an example, inactive recombinant TGase fusion proteins werepurified in a denaturing solution, refolded, and stored at aconcentration of about at least 1 mg/ml in a storage buffer, whichincludes about 50 mM of Tris-HCl (at a pH of about 8.0), about 50 mM ofKCl, about 0.1 mM of EDTA, and about 50% of glycerol. Another example ofstorage buffer includes about 50 mM of Tris-acetic acid (at a pH ofabout 6.0), about 50 mM of KCl, about 0.1 mM of EDTA, and about 50% ofglycerol.

[0091] As another examples, compounds to be cross-linked can bedenatured, refolded, and stored in various buffer solutions forlong-term storage and changing their specificity and accessibility offunctional groups toward the biological agent chosen duringcross-linking reaction. Exemplary compounds to be cross-linked thatexhibit change of reactivity to the chosen biological agent throughafter such procedures include, but are not limited to, bovine serumalbumin (BSA), histone H3 protein, glucose oxidase, ovalbumin, myelinbasic protein (MBP), recombinant serum albumin, recombinant cellulase,recombinant bovine serum albumin (BSA), recombinant tumour necrosisfactors (TNF-α), and recombinant epidermal growth factor receptor(EGF-R). Accordingly, the purification, inactivating, storing, and/orreactivating methods as decribed herein can be employed to othercompounds to be cross-linked as well.

[0092] Once the biological agents have been purified, inactivated andstored, they are later reactivated for use as cross-linking agents. Inone embodiment, the purified recombinant biological agents aredenatured, refolded, and inactivated and reactivated such that theirsubstrate specificity are altered, modified, or extended to react withand cross-link a broader range of compounds than their native biologicalgaent counterparts. Examples include the purified recombinant SM TGaseand SC TGase fusion proteins, however, the methods described herein canbe employed to other biological agents as well.

[0093] Another embodiment of the invention provides an activationsolution including at least one reducing agent, deionized water, and apH-buffering agent for adjusting the pH. Exemplary reducing agentsinclude dithiothreitol (DTT), glutathione, and the like, at aconcentration up to about 0.5 M. In another embodiment, the activationsolution further includes up to about 70% of glycerol. The pH of theactivation solution can be about pH 5 to about pH 11, such as betweenabout pH 6 to about pH 9. The activation solution can further includephosphate-buffered saline solution (PBS). One formulation of theactivation solution includes about 10 mM DTT, about 20% to about 30% ofglycerol, about 50 mM Tris buffer, and tritated with hydrochloric acidto a pH of about 7.4. Another formulation includes PBS solution, about30% of glycerol, about 50 mM Tris buffer, titrated with acetic acid to apH of about 6. In another formulation (discussed in the next section),the activation solution is the same solution that is used for thecross-linking reaction. For example, the invention provides a method ofactivating the TGases and cross-linking a compound in a single step.

[0094] For example, the activity of a recombinant transglutaminasepurified and inactivated by methods of the present invention wasassayed. The unit of transglutaminase activity was defined by the methodof the Folk and Cole (J. Biol. Chem., vol 241, p. 5518 (1966)) activityassay. Through repeated experimentation, it was found that the activityof the recombinant transglutaminases could be restored only when thepurified recombinant transglutaminases intentionally were kept in aninactive form in the storage buffer. The enzymatic activity of theinactivated recombinant transglutaminase fusion proteins was restored bythe addition of an activation solution, which included at least areducing agent and a pH-buffering agent for adjusting the pH of thecomposition. For example, a good reducing agent found to restore theenzymatic activity of recombinant transglutaminase fusion proteins wasdithiothreitol (DTT), at a concentration up to about 0.5 M. However,other reducing agents, such as glutathione and others, may also be used.The enzymatic activity of the active recombinant transglutaminase is atleast about 0.5 unit/mg in the presence of the activation solution, suchas about 1 unit/mg when about 0.005 unit per 1 mg of β-casein substratewas used in the activity assay. Also, it was found that the addition ofglycerol helped to activate the activity of the recombinanttransglutaminase fusion proteins, probably by stablizing the refoldedstructure of the recombinant transglutaminase fusion proteins.

[0095] Surprisingly, there was a change of solution color from clear toyellow when the recombinant transglutaminases were activated. The changeof solution color was observed for both the purified recombinant SMTGase fusion protein and the purified recombinant SC TGase fusionprotein. The results were measured as an increase in absorbance valuefrom 400 nm to 500 nm from about 0.0001 to about 0.1 or more, such as anincreased absorbance value of about 0.1 or more from 400 nm to 500 nm.For example, the solution includes an increased absorbance value ofabout 0.1 or more at OD₄₅₀, such as about 0.2 or more and as much asabout 2.0 or more. For example, in the presence of the activationsolution, there was an increase in OD₄₅₀ value for the solution of thepurified recombinant SM TGase and SC TGase fusion proteins, such as anincreased OD₄₅₀ value from about 0.1 or less to about 0.1 or more whenare of the purified recombinant SM TGase and SC TGase fusion proteinsactivated, e.g., OD₄₅₀ value at about 0.3 or more in the presence of oneactivation solution and OD₄₅₀ value at about 2.0 or more in the presenceof another activation solution.

[0096] Furthermore, the enzymatic activity was found to work at a widerange of temperature and, surprisingly, the activity was found optimalat room temperature as compared to high temperature, such as 37° C. Theresult is unexpected, as most microbial Ca²⁺-independenttransglutaminases have higher enzymatic activity at a temperature of 30°C. or more (see U.S. Pat. No. 6,100,053 and EP-0,481,504).

[0097] The purified recombinants SM TGase and SC TGase fusion proteinsexhibit enzymatic activity at broad pH optimum, from about 5 to about11, such as about 5.5 to about 9. Further, it was found that therecombinant SM TGase fusion protein exhibit higher cross-linkingabtility at pH of about 6 as compared to higher pH that is verydifferent from native SM TGase and from other microbialtransglutaminases which exhibit higher enzymatic activity only atneutral or higher pH (U.S. Pat. No. 6,100,053 and WO 00/70026).

[0098] One advantage of storing recombinant transglutaminases in aninactive form is that, after reactivation, the initial level ofenzymatic activity is not decreased. Native transglutaminases, whenpurified, usually precipitate out of solution into white proteinaggregates when purified. Additionally, native TGases sometimes reactwith their own protein species to form cross-linked transglutaminases.As a result, enzymatic activity is lost over time. However, an evengreater benefit of the inactivation/activation reaction has beendiscovered. Native transglutaminases react only with a limited number ofsubstrates, such as casein and other crude protein mixtures. For thesereasons, cross-linking applications for native transglutaminases isextremely limited by substrate specificity. A large number of proteins,such as bovine serum albumin (BSA), glucose oxidase, and ovalbumin, aswell as most peptides, do not react with native transglutaminases.However, the reversibly inactive biological agents described hereinreact with a large number of compounds after purification, inactivationand activation. The methods described herein decrease the fidelity ofthe TGases for substrates, allowing reaction with candidate compoundsincluding, but not limited to, bovine serum albumin (BSA), histone H3protein, tumour necrosis factors (TNF-α and other TNFs), glucoseoxidase, epidermal growth factor receptor (EGF-R), ovalbumin, and myelinbasic protein (MBP), as well as most naturally occurring peptides, andsynthetie peptides having at least one glutamine residue.

[0099] V. Compounds to be Cross-linked

[0100] Candidate compounds to be cross-linked are obtained from a widevariety of sources including combinatorial libraries of synthetic ornatural compounds. For example, numerous means are available for randomand directed synthesis of a wide variety of organic compounds, proteinsor peptides, and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare readily available or can be produced. Moreoever, naturally orsynthetically produced compounds are readily modified throughconventional chemical, physical and biochemical means, and may be usedto produce combinatorial libraries. Known pharmacological chemicals maybe subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0101] In addition to recombinant production, the whole compounds orfragments of compounds to be cross-linked may be produced by directpeptide synthesis using solid-phase techniques (Merrifield, J. (1963) J.Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using an Applied Biosystems 431A peptide sythesizer (PerkinElmer). Various fragments of compounds to be cross-linked may bechemically synthesized separately and combined using chemical methods toproduce the full-length molecule.

[0102] At least two types of candidate compounds or synthetic peptides,as representatives of compounds to be cross-linked by a biologicalagent, may be used. One type includes internal residues or functionalgroups that are reactive to a biological agent and can be cross-linkedby the biological agent of the invention. The other type includes anaddition of one or more terminal residues or functional groups to bereactive to a biological agent. For example, the presence or addition ofat least one isodityrosine residue to candidate compounds is requiredfor cross-linking reactions mediated by peroxidase/ascorbate oxidase(Cooper et al., 1983). As another example, the presence or addition ofhydroxylysine or lysine residue in candidate compounds is necessary forcross-linking reactions carried out by lysyl oxidases through oxidativedeamination of these reactive residues (Palamakambura et al., 2002).

[0103] However, when a biological agent, such as peroxidases are used, anumber of amino acid residues and derivatives thereof may serve as thereactive residues for forming the cross-linked bonds and generallyrequire the presence of peroxide (H₂O₂) in addition to the biologicalagent (the peroxidases, in this case) and the candidate compounds to thecross-linked (Otte et al, 2000; Fu et al., 2002).

[0104] As an example, candidate compounds in the reaction mixture of thecross-linking may include one or more candidate protein or peptide thatmay be expressed or otherwise present in a host cell. For example,candidate compounds include polyamino acids, cell-membrane-associatedproteins, tumor-associated antigens, cytokines, cytokine receptors,bacterial toxins, whole bacterial cells, viral coat proteins, wholeviruses, viral glycoproteins, cell wall-derived coat proteins, peptides,synthetic peptides, any modification of the aforementioned compounds,and derivatives thereof; and each candidate compound may be one or moremembers of library of proteins or peptides, such as a collection ofhuman ESTs, a total library of human ESTs, a collection of domainstructures (e.g. Zn-finger protein domains), or a totally random peptidelibrary.

[0105] As another example, the candidate compounds in the mixture of thecross-linking may include one or more antigens, e.g., disease-associatedantigens, cancer-specific antigens, and cancer-associated antigens, andcombinations thereof. Exemplary antigens include tumor surface antigensamong others, such as B-cell idiotypes, CD20 on malignant B cells, CD33on leukemic blasts, and HER2/neu on breast cancer. Other examplesinclude oncogenes or mutated tumor suppressor genes that have lost itstumor-suppressing function and may render the cells more susceptible tocancer. Tumor suppressor genes are genes that function to inhibit thecell growth and division cycles, thus preventing the development ofneoplasia. Mutations in tumor suppressor genes cause the cell to ignoreone or more of the components of the network of inhibitory signals,overcoming the cell cycle check points and resulting in a higher rate ofcontrolled cell growth—cancer. Examples of the tumor suppressor genesthat can be used include, but are not limited to, DPC-4, NF-1, NF-2, RB,p53, WT1, BRCA1 and BRCA2. DPC-4 is involved in pancreatic cancer andparticipates in a cytoplasmic pathway that inhibits cell division. NF-1codes for a protein that inhibits Ras, a cytoplasmic inhibitory protein.NF-1 is involved in neurofibroma and pheochromocytomas of the nervoussystem and myeloid leukemia. NF-2 encodes a nuclear protein that isinvolved in meningioma, schwanoma, and ependymoma of the nervous system.RB codes for the pRB protein, a nuclear protein that is a majorinhibitor of cell cycle. RB is involved in retinoblastoma as well asbone, bladder, small cell lung and breast cancer. p53 codes for p53protein that regulates cell division and can induce apoptosis. Mutationand/or inaction of p53 is found in a wide ranges of cancers. WT1 isinvolved in Wilms tumor of the kidneys. BRCA1 is involved in breast andovarian cancer, and BRCA2 is involved in breast cancer.

[0106] Other candidate compounds to be cross-linked include, but are notlimited to, cytokines, cytokine receptors, growth factor receptors andcombinations thereof. Exemplary growth factors include, but are notlimited to, epidermal growth factors (EGFs), transferrin, insulin-likegrowth factor, transforming growth factors (TGFs), interleukin-1, andinterleukin-2. The candidate compounds may also be one or more cellsurface proteins or receptors, such as various matrix metalloproteases,receptors associated with coronary artery disease, e.g. plateletglycoprotein lib/IIIa receptor, with autoimmune diseases such as CD4,CAMPATH-1 and surface components of the bacterial cell wall. As anotherexamples, the candidate compounds may also be one or more proteins orpeptides associated with human immune and/or allergic diseases, such asthose inflammatory mediator proteins, and peptides and proteins derivedfrom HLA class I and class 11 peptides, auto-antigens, e.g.Interleukin-1 (IL-1), tumor necrosis factor (TNF), leukotriene receptorand 5-lipoxygenase, and adhesion molecules such as VCAM-1 and VCAMNLA4.In addition, IgE may also serve as the candidate antigen because IgEplays pivotal role in type I immediate hypersensitive allergic reactionssuch as asthma.

[0107] Further, the candidate compounds may also be a viral surface orcore protein which may serve as an antigen to trigger immune response ofthe host. Examples of these viral proteins include, but are not limitedto, glycoproteins (or surface antigens, e.g., GP120 and GP41) and capsidproteins (or structural proteins, e.g., P24 protein); surface antigensor core proteins of hepatitis A, B, C, D or E virus (e.g. smallhepatitis B surface antigen (SHBsAg) of hepatitis B virus and the coreproteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein(G-protein) or the fusion protein (F-protein) of respiratory syncytialvirus (RSV); surface and core proteins of herpes simplex virus HSV-1 andHSV-2 (e.g., glycoprotein D from HSV-2).

[0108] Advantageously, a mixture of one or more candidate compounds asdescribed herein can be cross-linked by the biological agents of theinvention to generate high potency polyvalent antigens. For example, amixture of two or more candidate compounds was successfully used forgenerating cross-linked products for immunizing animals such as mice,rats, rabbits and others. The antibodies or antisera obtained from usingcross-linked products generated by the methods of the invention revealedan increased titer to each component of the mixture (each candidatecompound) than antibodies or antisera obtained from using conventionalnon-cross-linked antigens. For example, the titer of the antisera hasbeen found at least about 1000 or more to each component of thecandidate compounds chosen and the difference has been found to be atleast two fold higher titer, as much as about 30 fold or higher, and insome cases, about 80 fold or higher.

[0109] VI. Peptide Compositions

[0110] As discussed, many compounds can be used for cross-linking, butin one embodiment, such compounds have a peptide component. Peptides tobe used for cross-linking may be produced by recombinant means or may bechemically synthesized by, for example, the stepwise addition of one ormore amino acid residues in defined order using solid phase peptidesynthetic techniques. The peptides may need to be synthesized incombination with other proteins and then subsequently isolated bychemical cleavage. For example, short chain peptides can be synthesizedusing an automatic peptide synthesizer. Alternatively, different shortchain peptide species can be obtained from a long polypeptide chain,whether naturally-occurring or synthetic, through enzymatic reactionsand other means, and by purification of different peptide species usingcolumn chromatography.

[0111] In one embodiment, the functional groups for reacting to a chosenbiological agent, e.g., lysine and glutamine residues fortransglutaminases, may be located internally, i.e., not at the peptidetermini, within the peptide chain. Such a peptide monomer having alength of about 100 or less amino acids typically is a weak antigen forstimulating immune responses in animals.

[0112] As an example, peptides having at least one lysine (K) and atleast one glutamine (Q) residue are prepared to be cross-linked. Oneexample of a peptide having internal reactive glutamine and lysineresidues is the β-amyloid peptide. An exemplary synthetic β-amyloidpeptide (SEQ ID NO. 15) is provided herein. One example of a biologicalagent used herein is a purified recombinant transglutaminase fromStreptoverticillium mobaraense (ATCC 29032). By incubating the exemplarysynthetic peptides with the purified recombinant microbialtransglutaminase, a γ-glutamyl-ε-lysyl crosslinking/bridging bond isformed between the lysine and glutamine residues.

[0113] As a result of the activity of the biological agent, cross-linkedpeptides having a length of at least two peptide monomers are formed.The length of the resulting cross-linked peptides may be about 100 aminoacids or more, and up to about 1000 amino acids or more. Thecross-linked peptides can be used as antigens for stimulating immuneresponses in animals. In general, cross-linked antigens can inducehigher immune responses than monomeric antigens.

[0114] In an alternative embodiment, peptide monomers are synthesizedthat have reactive residues or functional groups on one or both termini.For example, when transglutaminase is chosen as the biological agent,the sequence of each monomer may vary as long as each monomer has oneore more glutamine (Q) residues on either one of the N-terminus or theC-terminus. Optionally, each monomer may have one or more lysine (K)residues. One example of a peptide having terminal reactive glutamineand lysine residues is a synthetic Bovine Serum Albumin peptide 5 (BSA5)having an amino acid sequence of SEQ ID NO. 16.

[0115] The peptide compositions of the invention may comprise naturallyoccurring amino acid residues or may contain non-naturally occurringamino acid residues such as certain D-isomers or chemically modifiednaturally occurring residues. These latter residues may be required, forexample, to facilitate or provide conformational constraints and/orlimitations to the peptides. The selection of a method of producing thesubject peptides depends on factors such as the required type, quantityand purity of the peptides as well as ease of production andconvenience.

[0116] The peptides prepared for cross-linking reaction may firstrequire chemical modification for use in vivo since the peptidesthemselves may not have a sufficiently long serum and/or tissuehalf-life. Chemical modification of the subject peptides may also beimportant to improve their antigenicity including the ability forcertain regions of the peptides to act as B and/or T cell epitopes. Suchchemically-modified synthetic peptides are referred to herein as“analogues”. The term “analogues” extends to any functional, chemical,or recombinant equivalent of the peptides of the present inventioncharacterized, in one embodiment, by their possession of at least one Bcell epitope. The term “analogue” is also used herein to extend to anyamino acid derivative of the peptides as described above. Analogues ofthe synthetic peptides contemplated herein include, but are not limitedto, peptides with modifications to their side chains, peptides withunnatural amino acids and/or their derivatives or other moleculesincorporated during peptide synthesis, and peptides treated withcross-linking agents or other agents which impose conformationalconstraints on the peptides or their analogues.

[0117] Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation (e.g. reaction with an aldehyde followed by reduction withSodium borohydride (NaBH₄), amidination with methylacetimidate,acylation with acetic anhydride, carbamoylation of amino groups withcyanate, trinitrobenzylation of amino groups with 2, 4,6-trinitrobenzene sulphonic acid (TNBS), acylation of amino groups withsuccinic anhydride and tetrahydrophthalic anhydride, and pyridoxylationof lysine with pyridoxal-5′-phosphate followed by reduction with sodiumborohydride (NaBH₄). In addition, the guanidine group of arginineresidues may be modified by the formation of heterocyclic condensationproducts with reagents such as 2,3-butanedione, phenylglyoxal andglyoxal.

[0118] The carboxyl group of side chains of peptides may be modified bycarbodiimide activation via O-acylisourea formation followed bysubsequent derivitisation, for example, to a corresponding amide.Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

[0119] Tryptophan residues may be modified by, for example, oxidationwith N-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

[0120] Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

[0121] Examples of incorporating unnatural amino acids and derivativesduring peptide synthesis include, but are not limited to, use ofnorleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoicacid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

[0122] VII. Cross-Linking a Compound by Transglutaminase

[0123] The present invention also provides methods for cross-linking acompound using a transglutaminase. FIG. 2 depicts such a method 200. Atstep 210, at least one compound having one or more glutamine (Q)residues is prepared. Glutamine residue is provided as the acyl donorfor transglutaminase-mediated cross-linking reaction. Typically, anycompound having an amino group can be used as the acyl receptor fortransglutaminase activity. Optionally, the compound further includes oneor more lysine (K) residues to be used as acyl receptors. In some cases,one compound having one or more glutamine (Q) residues on one terminusand one or more lysine (K) residues on the other terminus is prepared.For example, the lysine and glutamine residues as illustrated at step210 are either located internally—i.e., not at the termini but withinthe polypeptide chain—and/or terminally.

[0124] At step 220, the at least one compound is cross-linked by abiological agent, such as a transglutaminase, and cross-linked productshaving a size of at least two compound monomers are formed at step 230.For example, by incubating the compound with a purified recombinantmicrobial transglutaminase in the presence of an activation solutionunder suitable conditions, a y-glutamyl-E-lysyl crosslinking/bridgingbond is intermolecularly or intramolecularly formed between the lysineand glutamine residues. As an example, a number of native proteins,recombinant proteins, in purified or crude forms, are substrates ormodified to be substrates, as described herein using methods of theinvention and can be cross-linked accordingly. Exemplarytransglutaminase-reactive substrates that are cross-linked by thepurified recombinant transglutaminase include various plant proteins andanimal proteins, such as β-casein, β-lactoglobulin, ovalbumin, myosin,actin, serum albumin, gelatin, collagen, etc., and combinations thereof.Exemplary non-substrates that can be modified to be cross-linked by thepurified recombinant tranglutaminase include, but are not limited to,recombinant bovine serum albumin (BSA), histone proteins, glucoseoxidase, recombinant tumour necrosis factors, myelin basic protein(MBP), recombinant epidermal growth factor receptor (EGF-R), recombinantserum albumin, recombinant cellulase, and combinations or derivativesthereof.

[0125]FIG. 2 illustrates the use of a transglutaminase; however otherbiological agents may be employed, such as transferases,oxidoreductases, and the like. Reaction conditions for cross-linking ofcompounds by other biological agents will vary depending on the agents,the compounds, the volume of the reaction and the concentration andreactivity of the reactants. The cross-linked products can be checked orvisualized on a standard SDS-PAGE gel or other means to show thecompletion of the cross-linking reaction.

[0126] VIII. Cross-Linked Compounds as Therapeutics

[0127]FIG. 3 is a flow chart 300 illustrating the uses and theapplications of cross-linked products. At step 310, candidate compoundsare prepared or synthesized. At step 320, the compounds are cross-linkedby a biological agent, such as a purified recombinant biological agent,e.g., a purified recombinant transglutaminase from Streptoverticilliummobaraense (ATCC 29032).

[0128] At step 330, cross-linked products having a length of at leasttwo compound monomers are formed and obtained. The cross-linked productscan be used as polyvalent antigens for stimulating immune responses inanimals. The invention provides evidence that polyvalent antigens usingthe cross-linked products of the invention can induce increased immuneresponses than monomeric antigens. The in vivo results obtained havedemonstrated an increase in immune response for polyvalent antigensprepared according to the methods of the invention than conventionalnon-cross-linked antigens (See Experimental). For example, the titer ofthe antisera has been found at least about 1000 or more to eachmonomeric component of the candidate compounds chosen. In addition, theantibodies or antisera obtained exhibited an increased titer of about atleast 10 fold or higher, such as about 30 fold or higher, as much asabout 80 fold or higher.

[0129] At step 340, the cross-linked products are used directly astherapeutic agents to be administered into animals for treating diseasesassociated with the compound monomer. The therapeutic agents andvaccines of the present invention are used to induce acquired immunitythrough both active immunity and passive immunity. Such immunotherapyapplication can be tested in animal models before administration tohumans. In addition, the cross-linked products, polyvalent antigens, andantibodies of the invention are used in diagnostic kits for variousdiseases associated with the biological agents and candidate compoundsof the invention.

[0130] At step 350 the cross-linked products are used to produceantibodies in animals. The antibody produced is then used for developingvaccines and diagnostic kits at step 360. Thus, the polyvalent antigensusing cross-linked products of the present invention can be used astherapeutic agents or vaccines directly, or used as antigens to elicitantibodies in an animal, where the antibodies are then used astherapeutic agents or vaccines.

[0131] For example, when candidate compounds such as disease-associatedantigens, cancer-specific antigens, and cancer-associated antigens arecross-linked by the methods of the invention, the resulting cross-linkedproducts can be used as polyvalent antigens for direct immunization toinduce immune response in animals and treat the associated diseases orcancers. In addition, antibodies selected against these antigens can beused in a wide variety of therapeutic and diagnostic applications, suchas treatment of cancer by direct administration of the antibody alone(e.g., humanized antibodies for immunizing humans) or conjugated with aradioisotope or cytotoxic drug, or in a combination therapy involvingco-administration of cross-linked polyvalent antigens or antibodythereof with a chemotherapeutic agent, or in conjunction with radiationtherapy.

[0132] Therapeutics and Vaccines in General

[0133] For immunizing the polyvalent antigens of the invention and forthe production of antibodies, various hosts including goats, rabbits,rats, mice, humans, and others, may be immunized by injection with thecross-linked products obtained or fragments or oligopeptides thereofthat have immunogenic properties. Depending on the host species, variousadjuvants may be used to increase immunological response. Such adjuvantsinclude, but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin), aluminum hydroxide, and Corynebacterium parvum areespecially preferable. Other immune response enhancing compounds includeconjugate compound, co-stimulating factor for immune response, DNAvaccine, and combinations thereof. The above mentioned immune responseenhancing compounds can be formulated and immunized together with thevaccine and therapeutics of the invention or as one or more boosts forstimulating immune response after the vaccine and therapeutics of theinvention has been used as the vaccine.

[0134] The vaccines and therapeutics of the present invention can beadministered by oral, pulmonary, nasal, aural, anal, dermal, ocular,intravenous, intramuscular, intraarterial, intraperitoneal, mucosal,sublingual, subcutaneous, or intracranial route. In pharmaceutical,personal care, or veterinary applications, the vaccines and therapeuticsdescribed herein may be topically administered to any epithelialsurface. Such epithelial surfaces include oral, ocular, aural, anal andnasal surfaces, to treat, protect, repair or detoxify the area to whichthey are applied.

[0135] The therapeutics and vaccines of the invention can beincorporated into a variety of formulations for therapeuticadministration. Particularly, compounds, cross-linked products,biological agents, and polyvalent antigens that modulate the activity ofone or more disease-related proteins are formulated for administrationto patients for the treatment of disease. More particularly, thecross-linked products, biological agents, polyvalent antigens andcompounds of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants, gels, microspheres, and aerosols. As such,administration can be achieved in various ways, including oral, buccal,rectal, parenteral, intraperitoneal, intradermal, transdermal,intracheal, etc., administration.

[0136] The therapeutics and vaccines of the invention may be systemicafter administration or may be localized by the use of an implant or asurface patch that acts to retain the active dose at the site ofimplantation or contact. Implants for sustained release formulations arewell-known in the art. Implants are formulated as microspheres, slabs,etc. with biodegradable or non-biodegradable polymers. For example,polymers of lactic acid and/or glycolic acid form an erodible polymerthat is well-tolerated by the host. The implant is placed in proximityto the site of infection, so that the local concentration of activeagent is increased relative to the rest of the body.

[0137] The therapeutics and vaccines of the present invention can beadministered alone, in combination with each other, or they can be usedin combination with other known compounds. In pharmaceutical dosageforms, the therapeutics and vaccines may be administered in the form oftheir pharmaceutically acceptable salts, or they may also be used aloneor in appropriate association, as well as in combination with otherpharmaceutically active compounds.

[0138] For oral preparations, the therapeutics and vaccines can be usedalone or in combination with appropriate additives to make tablets,powders, granules or capsules, for example, with conventional additives,such as lactose, mannitol, corn starch or potato starch; with binders,such as crystalline cellulose, cellulose derivatives, acacia, cornstarch or gelatins; with disintegrators, such as corn starch, potatostarch or sodium carboxymethylcellulose; with lubricants, such as talcor magnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0139] Alternatively, the therapeutics and vaccines can be formulatedinto preparations for injections by dissolving, suspending oremulsifying them in an aqueous or nonaqueous solvent, such as vegetableor other similar oils, synthetic aliphatic acid glycerides, esters ofhigher aliphatic acids or propylene glycol; and if desired, withconventional additives such as solubilizers, isotonic agents, suspendingagents, emulsifying agents, stabilizers and preservatives.

[0140] The therapeutics and vaccines can be utilized in aerosolformulation to be administered via inhalation. The therapeutics andvaccines of the present invention can be formulated into pressurizedacceptable propellants such as dichlorodifluoromethane, propane,nitrogen and the like.

[0141] Furthermore, the therapeutics and vaccines can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The therapeutics and vaccines of thepresent invention can be administered rectally via a suppository. Thesuppository can include vehicles such as cocoa butter, carbowaxes andpolyethylene glycols, which melt at body temperature, yet are solidifiedat room temperature.

[0142] Pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public andknown in the art. Further, pharmaceutically acceptable auxiliarysubstances, such as pH adjusting and buffering agents, tonicityadjusting agents, stabilizers, wetting agents and the like, are readilyavailable to the public and known in the art.

[0143] The use of liposomes as a delivery vehicle is one method ofinterest. The liposomes fuse with the cells of the target site anddeliver the contents of the lumen intracellularly. The liposomes aremaintained in contact with the cells for sufficient time for fusion,using various means to maintain contact, such as isolation, bindingagents, and the like. In one aspect of the invention, liposomes may beaerosolized for pulmonary administration. Liposomes may be prepared withpurified proteins or peptides that mediate fusion of membranes, such asSendai virus or influenza virus, etc. The lipids may be any usefulcombination of known liposome forming lipids, including cationic lipids,such as phosphatidylcholine. The remaining lipid will normally beneutral lipids, such as cholesterol, phosphatidyl serine, phosphatidylglycerol, and the like. For preparing the liposomes, the proceduredescribed by Kato et al. (1991) J. Biol. Chem. 266:3361 may be used.Briefly, the lipids and lumen composition containing the nucleic acidsare combined in an appropriate aqueous medium, conveniently a salinemedium where the total solids will be in the range of about 1-10 weightpercent. After intense agitation for short periods of time, from about5-60 sec., the tube is placed in a warm water bath, from about 25° C. toabout 40° C. and this cycle repeated from about 5 to 10 times. Thecomposition is then sonicated for a convenient period of time, generallyfrom about 1-10 sec. and may be further agitated by vortexing. Thevolume is then expanded by adding aqueous medium, generally increasingthe volume by about from 1-2 fold, followed by shaking and cooling. Thismethod allows for the incorporation into the lumen of high molecularweight molecules.

[0144] The exact dosage of the chosen formulation for the chosen methodof administration will be determined by the medical practitioner, inlight of factors related to the subject that requires treatment. Dosageand administration are adjusted to provide sufficient levels of theactive moiety or to maintain the desired effect. Factors which may betaken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0145] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of therapeutics and vaccines will be specific toparticular cells, conditions, locations, etc.

[0146] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier. The term “unit dosage form,” asused herein, refers to physically discrete units suitable as unitarydosages for human and animal subjects, each unit containing apredetermined quantity of therapeutics and vaccines of the presentinvention calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for the novel unit dosage formsof the present invention depend on the particular compound employed andthe effect to be achieved, and the pharmacodynamics associated with eachcompound in the host.

[0147] A therapeutically effective dose refers to that amount of activeingredient—for example, cross-linked products or antibodies thereof,which ameliorates one or more symptoms or conditions. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio of toxic totherapeutic effects is the therapeutic index, which can expressed as theratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

[0148] Those of skill will readily appreciate that dose levels can varyas a function of the specific compound or therapeutics, the severity ofthe symptoms and the susceptibility of the subject to side effects. Someof the specific therapeutics are more potent than others. Preferreddosages for a given compound or therapeutic agent are readilydeterminable by those of skill in the art by a variety of means. Apreferred means is to measure the physiological potency of a giventherapeutic agent.

[0149] Cancer Immunotherapy

[0150] Methods of the invention can be used to cross-link a variety ofcompounds including, but not limited to, cytokines, cytokine receptors,growth factors, and growth factor receptors, to be adminstered inanimals to induce immune response and treat various diseases andcancers. As an example, one or more disease-associated antigens,cancer-specific antigens, and cancer-associated antigens, andcombinations thereof can be cross-linked by the biological agents of theinvention and obtained for formulating therapeutics and vaccine asdirect immunotherapy. As another example, high-level expression of EGFreceptor (EGF-R) can be found in a wide variety of human epithelialprimary tumors. Several murine monoclonal antibodies have beendemonstrated to be able to bind EGF receptors, block the binding ofligand to EGF receptors, and inhibit proliferation of a variety of humancancer cell lines in culture and in xenograft models (Mendelsohn andBaselga (1995) Antibodies to growth factors and receptors, in BiologicTherapy of Cancer, 2nd Ed., J B Lippincott, Philadelphia, pp607-623). Asanother example, TGF-α was found to mediate an autocrine stimulationpathway in cancer cells. Thus, polyvalent antigens and antibodiesselected against these cytokines and growth factors and generated usingthe method of the present invention can be used as a novel approach ofimmunotherapy.

[0151] As another example, polyvalent tumor suppressor antigens,oncogens, and/or antibodies derived therefrom, such as a mutated tumorsuppressor gene product, produced by using the method of the presentinvention can be used to intervene and block the interactions of thegene product with other proteins or biochemicals in the pathways oftumor onset and development.

[0152] Infectious Diseases

[0153] As another example, cell surface proteins, receptors, and surfacecomponents of an infectious agent (e.g., a bacteria, fungi, virus,algae, protozoan, and parasites, etc.) can be prepared by the methods ofthe invention to formulate therapeutics and vaccines for inducing activeand/or passive acquired immunity for treating or diagnosting theassociated diseases. For example, one or more viral glycoproteins and/orone or more infectious agents with or without being attenuated can becross-linked by the biological agents of the invention. The cross-linkedproducts can be formulated into therapeutics and vaccines for treatingor diagnosting the one or more diseases related to the virus andinfectious agents. This approach is very powerful for designingmultivalent vaccine and has the advantages of being able to treat morethan one diseases in a single vaccine formula.

[0154] Immune-Related and/or Autoimmune Diseases

[0155] As another example, for the treatment of patients with mycosisfungoides, generalized postular psoriasis, severe psorisis, andrheumatoid arthritis, antibodies against CD₄ has been tested in clinicaltrials. Further, antibodies against lipid-A region of the gram-negativebacterial lipopolysaccharide have been tested clinically in thetreatment of septic shock. As another example, antibodies againstCAMPATH-1 has also been tested clinically in the treatment againstrefractory rheumatoid arthritis (Vaswani et al. (1998) “Humanizedantibodies as potential therapeutic drugs” Annals of Allergy, Asthma andImmunology 81:105-115). Thus, polyvalent antigens and antibodiesselected against these cell surface molecules, cytokines, receptors, andgrowth factors generated by using the method of the present inventioncan be used to treat a variety of immune-related and/or autoimmunediseases.

[0156] As an example to proteins or peptides associated with humanimmune and/or allergic diseases, studies have shown that the level oftotal serum IgE tends to correlate with severity of such diseases,especially in asthma. Burrows et al. (1989) “Association of asthma withserum IgE levels and skin-test reactivity to allergens” New Engl. L.Med. 320:271-277. Thus, polyvalent IgE antigens and/or antibodiesselected against IgE prepared by using the method of the presentinvention may be used to reduce the level of IgE or block the binding ofIgE to mast cells and basophils in the treatment of allergic diseaseswithout having substantial impact on normal immune functions.

[0157] Uses of Cross-Linked Amyloid Peptides as Preventing orTherapeutic Agents against Alzheimer's Disease.

[0158] The invention also provides a method of using cross-linkedβ-amyloid peptides for preventing or as therapeutic agents forAlzheimer's disease. Embodiments of the invention pertain tocross-linked products, vaccines, compounds, and pharmaceuticalcompositions that bind to natural β-amyloid peptides, modulate theaggregation of natural β-amyloid peptides and/or inhibit theneurotoxicity of natural β-amyloid peptides (“modulator compounds”). Ithas recently been reported (Games et al. (1995) Nature 373:523-527) thatan Alzheimer-type neuropathology has been created in transgenic mice.The transgenic mice express high levels of human mutant amyloidprecursor protein and progressively develop many of the pathologicalconditions associated with Alzheimer's disease. Further, numerousstudies in humans also point to a direct pathological role for theβ-amyloid peptide in Alzheimer's diseases.

[0159] The β-amyloid modulator compounds of the invention comprise apeptidic structure and corss-linked products thereof prepared by themethods described herein. The peptide structure preferably based onβ-amyloid peptide, composed entirely of L- or D-amino acids. In variousembodiments, the peptidic structure of the modulator compound comprisescross-linked products of L- or D-amino acid sequences corresponding to aL-amino acid sequence found within natural β-amyloid peptide, or L- orD-amino acid sequences that are scrambled or substituted versions of thenatural β-amyloid peptide amino acid sequence. A D-amino acid sequenceis a retro-inverso isomer of a L-amino acid sequence. In addition, theL- or D-amino acid peptidic structure of the modulator can be designedbased upon a subregion of natural β-amyloid peptide.

[0160] For example, an amino acid sequence having lysine and glutamineresidues located internally within each amyloid peptide chain asillustrated in SEQ ID No. 15 was designed. A synthetic peptide monomerhaving a length of about 100 or less amino acids is a weak antigen forstimulating immune response in animals. However, such synthetic peptidesmay increase antigenic activity if they have been cross-linked by abiological agent. One example of biological agent used herein is apurified recombinant transglutaminase from Streptoverticilliummobaraense (ATCC 29032). By incubating the synthetic peptides with thepurified recombinant microbial transglutaminase, a γ-glutamyl-ε-lysylcrosslinking/bridging bond is intermolecularly formed between the lysineand glutamine residues.

[0161] A modulator drawn to this embodiment preferably includescross-linked products of 3-20 L- or D-amino acids, more preferably 3-10L- or D-amino acids and even more preferably 3-5 L- or D-amino acids.The peptidic structures of the modulator can have free amino- andcarboxy-termini. Alternatively, the amino-terminus, the carboxy-terminusor both may be modified. For example, an N-terminal modifying group canbe used that enhances the ability of the modulator to inhibit β-amyloidaggregation. Preferred amino-terminal modifying groups include cyclic,heterocyclic, polycyclic and branched alkyl groups. Examples of suitableamino-terminal modifying groups include cis-decalin-containing groups,biotin-containing groups, fluorescein-containing groups, adiethylene-triaminepentaacetyl group, a (−)-mentboxyacetyl group, anN-acetylneuraminyl group, a phenylacetyl group, a diphenylacetyl group,a triphenylacetyl group, an isobutanoyl group, a 4-methylvaleryl group,a 3-hydroxyphenylacetyl group, a 2-hydroxyphenylacetyl group, a3,5-dihydroxy-2-naphthoyl group, a 3,4-dihydroxycinnamoyl group, a(.+-.)-mandelyl group, a (.+-.)-mandelyl-(.+-.)-mandelyl group, aglycolyl group, a benzoylpropanoyl group and a 2,4-dihydroxybenzoylgroup. Moreover, the amino- and/or carboxy termini of the peptidemodulator can be modified to alter a pharmacokinetic property of themodulator (such as stability, bioavailability and the like). Preferredcarboxy-terminal modifying groups include amide groups, alkyl or arylamide groups (e.g., phenethylamide) and hydroxy groups (i.e., reductionproducts of peptide acids, resulting in peptide alcohols). Stillfurther, a modulator compound can be modified to label the modulatorwith a detectable substance (e.g., a radioactive label).

[0162] The modulators of the invention can promote amyloid aggregationor, more preferably, can inhibit natural amyloid aggregation. In apreferred embodiment, the cross-linked modulator compounds modulate theaggregation of natural β-amyloid peptides (β-AP). In a preferredembodiment, the β-amyloid modulator compounds of the invention arecomprised of β-amyloid aggregation core domain and a modifying groupcoupled thereto such that the modulator alters the aggregation orinhibits the neurotoxicity of natural β-amyloid peptides when contactedwith the peptides. Furthermore, the modulators are capable of alteringnatural β-amyloid peptide aggregation when the natural β-amyloidpeptides are in a molar excess amount relative to the modulators.Pharmaceutical compositions comprising the modulators of the invention,and diagnostic and treatment methods for amyloidogenic diseases usingthe modulators of the invention, are also disclosed.

[0163] This invention pertains to cross-linked products, modulatorcompounds, and pharmaceutical compositions thereof, that can modulatethe aggregation of amyloidogenic proteins and peptides, in particulartherapeutic and vaccines that can modulate the aggregation of naturalβ-amyloid peptides and inhibit the neurotoxicity of natural β-amyloidpeptides. In one embodiment, the invention provides an amyloid modulatorcompound including amyloidogenic proteins, cross-linked productsthereof, or peptide fragments thereof, with or without coupling directlyor indirectly to one or more modifying groups. Preferably, the modulatorcompound modulates the aggregation of natural amyloid proteins orpeptides when contacted with the natural amyloidogenic proteins orpeptides. The amyloidogenic proteins, cross-linked products thereof, orpeptide fragments thereof, include, but are not limited to, naturalβ-amyloid peptides, transthyretin (TTR), prion protein (PrP), isletamyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa lightchain, lambda light chain, amyloid A, procalcitonin, cystatin C, β-2microglobulin, ApoA-I, gelsolin, calcitonin, fibrinogen, lysozyme, ndcombinations thereof.

[0164] Another aspect of the invention pertains to methods for treatinga subject for a disorder associated with P-amyloidosis. These methodsinclude administering to the subject a therapeutically effective amountof a modulator compound of the invention, such as cross-linked productsof peptides derived from SEQ ID No.15, such that the subject is treatedfor a disorder associated with β-amyloidosis. Preferably, the disorderis Alzheimer's disease.

[0165] Antibody Production

[0166] In adition to be used directly in formulating therapeutics andvaccines, the cross-linked products and biological agents of theinvention are useful for the production of antibodies. Antibodies may begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal antibodies, monoclonalantibodies, chimeric antibodies, humanized antibodies, neutralizingantibodies, single chain, Fab fragments, and fragments produced by Fabexpression libraries.

[0167] Monoclonal antibodies may be prepared using any technique thatprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique; mouse, rabbit, andother hybridoma techniques; and the EBV-hybridoma technique (Kohler, G.et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120).

[0168] In addition, techniques developed for the production of chimericantibodies involving the splicing of non-human antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceprotein-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0169] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299). Antibodyfragments containing specific binding sites for the cross-linkedpolyvalent antigens and biological agents of the invention may also begenerated. For example, such fragments include, but are not limited to,the F(ab′)₂ fragments which can be produced by pepsin digestion of theantibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, W. D. et al. (1989) Science 254:1275-1281).

[0170] Antibodies are prepared in accordance with conventional methods,where the cross-linked products, polyvalent antigens, and biologicalagents of the invention are used as an immunogen, by itself orconjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, otherviral or eukaryotic proteins, or the like. Various adjuvants may beemployed, with a series of injections, as appropriate. For monoclonalantibodies, after one or more booster injections, the spleen isisolated, the lymphocytes immortalized by cell fusion, and then screenedfor high affinity antibody binding. The immortalized cells, i.e.hybridomas, producing the desired antibodies may then be expanded. Forfurther description, see Monoclonal Antibodies: A Laboratory Manual,Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 1988. If desired, the mRNA encoding the heavy and lightchains may be isolated and mutagenized by cloning in E. coli, and theheavy and light chains mixed to further enhance the affinity of theantibody. Alternatives to in vivo immunization as a method of raisingantibodies include binding to phage display libraries, usually inconjunction with in vitro affinity maturation.

[0171] A variety of protocols are known in the art for detecting andmeasuring either polyclonal or monoclonal antibodies prepared by themethods of the invention and raised specifically for the variouscross-linked products, biological agents, and compounds of theinvention. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on the cross-linked products ispreferred, but a competitive binding assay may be employed. These andother assays are described, among other places, in Hampton, R. et al.(1990; Serological Methods, a Laboratory Manual, APS Press, St Paul,Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).Suitable reporter molecules or labels, which may be used to assaybinding or interaction of the antibody produced, includeradionucleotides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like. As an alternative to using cross-linked products to elicitantibodies, the cross-linked products may be administered alone, as apart of a pharmaceutical, personal care or veterinary preparation or aspart of a prophylactic preparation, administered by parenteral ornon-parenteral route.

[0172] Diagnostics Applications

[0173] The invention provides various antibodies raised against thecross-linked products, candidate compounds, and biological agents.Antibodies raised against these therapeutics and vaccines of theinvention may be used in staining or in immunoassays. Samples, as usedherein, include biological fluids such as semen, blood, cerebrospinalfluid, tears, saliva, lymph, dialysis fluid and the like; organ ortissue culture derived fluids; and fluids extracted from physiologicaltissues. Also included in the term are derivatives and fractions of suchfluids. The cells may be dissociated, in the case of solid tissues, ortissue sections may be analyzed. Alternatively a lysate of the cells maybe prepared.

[0174] Diagnosis may be performed by a number of methods to determinethe absence or presence or altered amounts of normal or abnormalantigens in patient cells. For example, detection may utilize stainingof cells or histological sections, performed in accordance withconventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies raised to the therapeutics and vaccines of thepresent invention are added to the cell sample, and incubated for aperiod of time sufficient to allow binding to the epitope, usually atleast about 10 minutes. The antibody may be labeled with radioisotopes,enzymes, fluorescers, chemiluminescers, or other labels for directdetection. Alternatively, a second stage antibody or reagent is used toamplify the signal. Such reagents are well known in the art. Forexample, the primary antibody may be conjugated to biotin, withhorseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to aflourescent compound, e.g. flourescein rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

[0175] In another embodiment, antibodies that specifically bind thetherapeutics and vaccines of the present invention may be used for thediagnosis of conditions or diseases characterized by expression of thecompounds, or in assays to monitor patients being treated with thecompounds themselves, agonists, antagonists, or inhibitors. Theantibodies useful for diagnostic purposes may be prepared in the samemanner as those described above for therapeutics. Diagnostic assays forthe therapeutics and vaccines include methods which utilize antibodiesraised to the cross-linked products, polyvalent antigens, candidatecompounds, and biological agents, and a label to detect compounds inhuman body fluids or extracts of cells or tissues. The antibodies may beused with or without modification, and may be labeled by joining them,either covalently or non-covanently, with a reporter molecule. A widevariety of reporter molecules which are known in the art may be used,several of which are described above.

[0176] A variety of protocols including ELISA, RIA, FACS for measuringantigens are known in the art and provide a basis for diagnosing alteredor abnormal levels of target protein expression. Normal or standardvalues for target protein expression are established by combining bodyfluids or cell extracts taken from normal mammalian subjects, preferablyhuman, with antibody to target protein under conditions suitable forcomplex formation. The amount of standard complex formation may bequantified by various methods, but preferably by photometric, means.Quantities of protein expressed in subject samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0177] Other Applications

[0178] The cross-linked products and biological agents of the inventionhave been shown to be useful for a variety of industrial purposes,including the field of processing of raw fish paste, tofu noodles,confectionery/bread, food adhesives, sheet-like meat food, yogurt,jelly, gelling of cheese proteins, for improving baking quality offlour, improving taste and texture of food proteins, as well as inleather processing (e.g. casein finishing), etc.

[0179] The cross-linked products produced by the biological agents ofthe invention can also be used as novel protein-derived materials in awide range of industries including cosmetics such as hair dyeingformulations for the production of keratinous fibre cross links, theproduction of thermally stable materials such as raw materials ofmicrocapsules, carriers of immobilized enzymes and the like.

EXPERIMENTAL

[0180] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention. Efforts have been madeto ensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Example 1 Isolation of Genomic DNA from Streptomyces mobaraensis andStreptomyces cinnamoneus

[0181] Cultures of Streptomyces mobaraensis (from an ATCC strain, No.29032) and Streptomyces cinnamoneus (from an ATCC Strain, No. 11874)were grown and harvested into cell pellets. The cell pellets were thenfreeze-dried before being resuspended and washed in double distilledwater and centrifuged again. The washed cell pellets were resuspended inlyses buffer (provided in the DNasey Tissue Kit from Qiagen, Inc.), andgenomic DNA was purified as described in a protocol of a DNasey TissueKit (from Qiagen, Inc.). Genomic DNA from both strains was purified tohomogeneity to be used as a Polymerase Chain Reaction (PCR) template forcloning of the microbial transglutaminase genes from Streptomycesmobaraensis and Streptomyces cinnamoneus.

EXAMPLE 2 Cloning of Transqlutaminase Genes

[0182] Cloning of transglutaminase genes from Streptomyces mobaraensisATCC 29032 (SM TGase) was accomplished by using purified genomic DNA asa PCR template in a PCR reaction and two primers as the 5′ and 3′primers, SEQ ID NO.1 and SEQ ID NO. 2, respectively. The sequences ofSEQ ID NO.1 and SEQ ID NO. 2 are based on the Gene bank Accession numberY18315 encoding the mature SM TGase polypeptide from Streptomycesmobaraensis DSMZ and further include pre-designed extended Nhe I andHind III recognition sequences respectively.

[0183] Cloning of transglutaminase genes from Streptomyces cinnamoneusATCC 11874 (SC TGase) was accomplished by using purified genomic DNA asa PCR template in a PCR reaction and two primers as the 5′ and 3′primers, SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The sequences ofSEQ ID NO. 3 and SEQ ID NO. 4 are based on the Gene bank Accessionnumber Y08820 encoding the mature SC TGase polypeptide from Streptomycescinnamoneus CBS 683.68 and further include pre-designed extended Nhe Iand Hind III recognition sequences, respectively.

[0184] PCR for cloning the SM TGase and SC TGase genes were performedfor 35 cycles, each cycle at about 94° C. for about 30 seconds, at about55° C. for about 45 seconds, and at about 68° C. for about 2 minutes,using the thermal enzyme, Advantage-2 DNA polymerase (Clontech). Afterthese cycles, Taq DNA polymerase (Invitrogen) was added to the PCRreactions and incubated at about 72° C. for about 15 minutes to addadenosine (A) overhangs at 3′ end of each PCR product.

[0185] The synthesized PCR products containing the SM TGase gene andpre-designed Nhe I and Hind III recognition sites were obtained andcloned into a vector, pCR2.1-TOPO (Invitrogen). Positive clones havingthe insert DNA of SM TGase gene, were sequenced and named aspCR2.1-SMTG. Likewise, the synthesized PCR products containing the SCTGase and pre-designed Nhe I and Hind III recognition sites wereobtained and cloned into a vector, pCR2.1-TOPO (Invitrogen). Positiveclones having the insert DNA of SC TGase gene, were sequenced and namedas pCR2.1-SCTG.

Example 3 DNA Sequences of Transqlutaminase Genes from Two G(+)Actinomycetes

[0186] Sequencing of pCR2.1-SMTG revealed a DNA sequence, SEQ ID No. 5,encoding the mature TGase protein from Streptomyces mobaraensis ATCC29032 without the signal peptide. The translated amino acid sequence isshown as SEQ ID No. 6.

[0187] Sequencing of pCR2.1-SMTG revealed a DNA sequence, SEQ ID No. 7,genes encoding the mature TGase protein from Streptomyces cinnamoneusATCC 11874 without the signal peptide. The translated amino acidsequence is shown as SEQ ID No. 8.

[0188] Table 1 is a comparison of the two DNA sequences SEQ ID No. 5(upper sequence) and SEQ ID No. 7 (lower sequence) by BLAST alignment.The alignment indicates about 84% sequence identity between the DNAsequence of the mature TGase protein from Streptomyces mobaraensis ATCC29032 and the mature TGase protein from Streptomyces cinnamoneus ATCC11874. TABLE 1 Blast alignment of SEQ ID No. 5 and SEQ ID No. 7.cagcagcggcctggtgccgcgcggcagccatatggctagc-cccga-----ctccgacga (SEQ IDNO.5) ||||||||||||||||||||||||||||||||||||||||||||      ||||||||cagcagcggcctggtgccgcgcggcagccatatggctagctcccgggccccctccgatga (SEQ IDNO.7.) cagggtcacccctcccgccgagccgctcgacaggatgcccgacccgtaccgtccctcgta||||  |||||||||||||||||||||||||||||||||  |||||||  |||ccgggaaactcctcccgccgagccgctcgacaggatgcctgaggcgtaccgggcctacggcggcagggccgagacggtcgtcaacaactacatacgcaagtggcagcaggtctacagcca |||||||||   ||||||||||||||||||||||||||||||||||||||||||||||aggcagggccactacggtcgtcaacaactacatacgcaagtggcagcaggtctacagtcaccgcgacggcaggaagcagcagatgaccgaggagcagcgggagtggctgtcctacggctg||||||||||||||||||||||||||||||||||||   ||||||||||||||ccgcgacggaaagaaacagcaaatgaccgaagagcagcgagaaaagctgtcctacggttgcgtcggtgtcacctgggtcaattcgggtcagtacccgacgaacagactggccttcgcgtc|||||||||||||||||||||||||  |||||||||||||||||||||||||||cgttggcgtcacctgggtcaactcgggcccctacccgacgaacagattggcgttcgcgtccttcgacgaggacaggttcaagaacgagctgaagaacggcaggccccggtccggcgagac|||||||||||||||||||||||||||||||||  ||||||||  |||  ||||cttcgacgagaacaagtacaagaacgacctgaagaacaccagcccccgacccgatgaaacgcgggcggagttcgagggccgcgtcgcgaaggagagcttcgacgaggagaagggcttcca|||||||||||||||||||||||||||||  ||||||||||||||||||||||gcgggcggagttcgagggtcgcatcgccaagggcagtttcgacgaggggaagggtttcaagcgggcgcgtgaggtggcgtccgtcatgaacagggccctggagaacgcccacgacgagag|||||||||||||||||||||||||||||||||||||||||||||||||||||||gcgggcgcgtgatgtggcgtccgtcatgaacaaggccctggaaaatgcccacgacgagggcgcttacctcgacaacctcaagaaggaactggcgaacggcaacgacgccctgcgcaacga  |||||||||||||||||||||||||  |||||  |||||||||||||  |||gacttacatcaacaacctcaagacggagctcacgaacaacaatgacgctctgctccgcgaggacgcccgttccccgttctactcggcgctgcggaacacgccgtccttcaaggagcggaa||||  |||||    ||||||||||||||||||||||||||||||||||||  |||ggacagccgctcgaacttctactcggcgctgaggaacacaccgtccttcaaggaaagggacggaggcaatcacgacccgtccaggatgaaggccgtcatctactcgaagcacttctggag||||||||  ||||||||||||||||||||||||||||||||||||||||||||||cggcggcaactacgacccgtccaagatgaaggcggtgatctactcgaagcacttctggagcggccaggaccggtcgagttcggccgacaagaggaagtacggcgacccggacgccttccg||||||||||||  ||||  ||||||||||||||||||||||||||||||||||||cgggcaggaccggcggggctcctccgacaagaggaagtacggcgacccggaagccttccgccccgccccgggcaccggcctggtcgacatgtcgagggacaggaacattccgcgcagccc|||||||||||||||||||||||||||||||||||||||||||||||||||||ccccgaccagggtaccggcctggtcgacatgtcgaaggacagaagcattccgcgcagtcccaccagccccggtgagggattcgtcaatttcgactacggctggttcggcgcccagacgga  |||  ||||||||||  |||||||||||||||||||||||||||||||||ggccaagcccggcgaaggttgggtcaatttcgactacggttggttcggggctcaaacagaagcggacgccgacaagaccgtctggacccacggaaatcactatcacgcgcccaatggcag|||||||||||||||||   |||||||||||  ||||||||||||||||||||agcggatgccgacaaaaccacatggacccacggcgaccactaccacgcgcccaatagcgacctgggtgccatgcatgtctacgagagcaagttccgcaactggtccgagggttactcgga||||||  |||||||||  ||||||||||||||||||||||||  |||||||||cctgggccccatgcacgtacacgagagcaagttccggaagtggtctgccgggtacgcggacttcgaccgcggagcctatgtgatcaccttcatccccaagagctggaacaccgcccccga|||||||||||||||||||||||||  ||||||||||||||||||||||||||||||cttcgaccgcggagcctacgtgatcacgttcatacccaagagctggaacaccgcccccgccaaggtaaagcagggctggccgtga ||||||  ||||||||||||||||caaggtggagcaaggctggccgtga

[0189] Table 2 is a comparison of the two amino acid sequences SEQ IDNo. 6 (upper sequence) and SEQ ID No. 8 (lower sequence) by BLASTalignment. The alignment indicates about 270 identical amino acids(middle sequence, about 81% sequence identity) between the amino acidsequence of the mature TGase protein (about 331 amino acids) fromStreptomyces mobaraensis ATCC 29032 and the amino acid sequence of themature TGase protein (about 334 amino acids) from Streptomycescinnamoneus ATCC 11874.

Example 4 Cloning of SM TGase Gene into an Expression Vector

[0190] The expression of the TGase genes in an expression vector has tobe tightly regulated. A pET expression vector (Studier et al., 1990) waschosen and combined with a 6×-histidine-tagged fusion protein system asa simplified purification scheme for the inducible expression ofrecombinant TGase protein. The pCR2.1-SMTG plasmid was digested with NheI and Hind Ill and the DNA fragment containing the SM TGase gene waspurified from the digest and subcloned into a pET-28a vector (Novagen).Positive clones with SM TGase gene insert, pET28-SMTG, were identifiedand sequenced.

[0191] Sequencing of pET28-SMTG reveals a DNA sequence, SEQ ID No. 9,encoding a recombinant 6×-His-TGase fusion protein of Streptomycesmobaraensis ATCC 29032. The translated amino acid sequence, SEQ ID No.10, encoding the recombinant 6×-His-TGase fusion protein of Streptomycesmobaraensis ATCC 29032 (about 355 amino acids) is similar to thesequence of a transglutaminase from Streptoverticillium spp. Strain-8112(Kanaji et al., 1994; Washizu et al., 1994; EP-A-0481 504). However, therecombinant TGase includes the extended 6×-His-tagged 24 amino acids inthe N-terminus (MGSSHHHHHHSSGLVPRGSHMASP-), which might help for therecombinant SM TGase fusion protein to fold properly in its structure.TABLE 2 Blast alignment of SEQ ID No. 6 and SEQ ID No. 8.   DSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGR (SEQ ID NO. 6)    SDDR TPPAEPLDRMP+YR   GRA TVVNNYIRKWQQVYSHRDG+SRAPSDDRETPPAEPLDRMPEAYRAYGGRATTVVNNYIRKWQQVYSHRDGK (SEQ ID NO. 8)KQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRP KQQMTEEQRELSYGCVGVTWVNSG YPTNRLAFASFDE+++KN+LKN  PKQQMTEEQREKLSYGCVGVTWVNSGPYPTNRLAFASFDENKYKNDLKNTSPRSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNLK R  ETRAEFEGR+AKSFDE KGF+RAR+VASVMN+ALENAHDE  Y++NLKRPDETRAEFEGRIAKGSFDEGKGFKRARDVASVMNKALENAHDEGTYINNLKKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKHFW  EL N NDAL  ED+RSFYSALRNTPSFKER+GGN+DPS+MKAVIYSKHFWTELTNNNDALLREDSRSNFYSALRNTPSFKERDGGNYDPSKMKAVIYSKHFWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFVNF SGQD+S+DKRKYGDP+AFRP  GTGLVDMS+DR+IPRSP  PGEG+VNFSGQDQRGSSDKRKYGDPEAFRPDQGTGLVDMSKDRSIPRSPAKPGEGWVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSEGYSD DYGWFGAQTEADADKTWTHG+HYHAPN  LG MHV+ESKFR WS GY+DDYGWFGAQTEADADKTTWTHGDHYHAPNSDLGPMHVHESKFRKWSAGYADFDRGAYVITFIPKSWNTAPDKVKQGWP* 331 amino acids FDRGAYVITFIPKSWNTAPKV+QGWP* FDRGAYVITFIPKSWNTAPAKVEQGWP* 334 amino acids

Example 5 Cloning of SC TGase Gene into an Expression Vector

[0192] The pCR2.1-SCTG plasmid was digested with Nhe I and Hind III andthe DNA fragment containing the SC TGase gene was purified from thedigest and subcloned into a pET-28a vector (Novagen). Positive cloneswith SC TGase gene insert, pET28-SCTG, were identified and sequenced.

[0193] Sequencing of pET28-SCTG reveals a DNA sequence, SEQ ID No. 11,genes encoding a recombinant 6×-His-TGase fusion protein fromStreptomyces cinnamoneus ATCC 11874. The translated amino acid sequence,SEQ ID No. 12, encoding the recombinant 6×-His-TGase fusion protein ofStreptomyces cinnamoneus ATCC 11874 (about 355 amino acids) is similarto the sequence of a transglutaminase from Streptomyces cinnamoneus CBS683.68 (Duran et al., 1998). They differ, however, in that therecombinant TGase includes the extended 6×-His-tagged 23 amino acids inthe N-terminus (MGSSHHHHHHSSGLVPRGSHMAS-), which might help therecombinant SC TGase fusion protein to fold properly and to be active.

Example 6 Over-Expression of Recombinant Transqlutaminases

[0194] In order to purify recombinant SM TGase and SC TGase, pET28-SMTGand pET28-SCTG were used to transform E. coli strain BL21 (DE3) cells(Novagen). Colonies with over-expression of N-terminus 6×His-tagged SMTGase fusion protein and N-terminus 6×His-tagged SC TGase fusion proteinwere screened. This was done by incubating each colony in about 1 ml LBmedium with added Kanamycin (about 50 μg/ml), adding about 1 mM of IPTGto each culture at OD600 of about 0.8 to induce the expression of thefusion proteins, and continuing the incubation for about 2 hours atabout 37° C. Clones with over-expression of recombinant transglutaminaseSM TGase and SC TGase were confirmed by Coomassie Blue staining ofSDS-PAGE gels. Induction of BL21(DE3)+pET28-SMTG E. coli cultures led tothe over-expression of a recombinant 6×His-tagged SM TGase fusionprotein and induction of BL21(DE3)+pET28-SCTG E. coli cultures led tothe over-expression of a recombinant 6×His-tagged SC TGase fusionprotein, as judged by Coomassie Blue staining of SDS-PAGE gels. Cellfractionation experiments indicated that the over-expressed fusionproteins were expressed as inclusion bodies inside the host E. colicell, instead of soluble cytoplasmic or secreted proteins.

Example 7 Purification of Recombinant Transqlutaminases in Single ColumnStep

[0195] For large-scale expression of recombinant transglutaminases, suchas SM TGase and SC TGase, an overnight culture (10 ml LB/Kanamycin) wasgrown at about 37° C. from a single colony of BL21(DE3)+pET28-SMTG orBL21(DE3)+pET28-SCTG. The 10 ml saturated overnight culture was added to250 ml LB/Kanamycin medium and incubated with shaking at 250 rpm atabout 37° C. for 4 hours until OD600 absorbance value of the culturereached at about 0.8. IPTG (about 1 mM final concentration) was added toinduce protein expression for 2 hours. After 2 hours, the induced cellswere harvested by centrifugation at 5,000×g, and cell pellet was frozenat about −80° C. for about 1 hour.

[0196] The frozen cell pellet was resuspended and lysed in about 10 mlof nickel Ni+binding buffer, containing about 6M guanidine titrated withhydrochloric acid (guanidine-HCl) to a pH of about 7.9, prepared in asolution containing about 20 mM of Tris buffer, about 5 mM imidazole,and about 0.5M sodium chloride (NaCl), in order to make all cellular andoverexpressed proteins denatured and soluable, before being centrifugedat 12,000×g for 30 minutes to spin down unlysed cell debris. Meanwhile,a His-Bind Ni column (Novagen) was inserted into a Vaccum manifold (BigBasin) and pre-wetted with about 15 ml of binding buffer run through thecolumn by applying a vacuum. After centrifugation, the supernatant wasloaded into the pre-wetted His-Bind Ni+ column and a vacuum was appliedto the column.

[0197] The loaded His-Bind Ni column was washed with 15 ml wash buffercontaining about 6 M guanidine titrated with hydrochloric acid to a pHof about 7.9, prepared in a solution containing about 20 mM of Trisbuffer, about 20 mM imidazole, and about 0.5M sodium chloride (NaCl).After washing, over-expressed recombinant fusion protein was eluted with10 ml of elution buffer containing about 6 M guanidine titrated withhydrochloric acid to a pH of about 7.9 and prepared in a solutioncontaining about 20 mM of Tris buffer, about 0.5M imidazole, and about0.5M sodium chloride (NaCl), and collected in a 15 ml Falcon tube. DTTwas added to the collected elution fraction to a final concentration ofabout 10 mM.

[0198] Because of the specificity of the Ni to the Hisx6 tag, thecollected elution fraction contains about 99% of denatured 6×His-taggedfusion proteins. The denatured fusion proteins were renatured and/orrefolded by adding to the collected elution fraction about 5 volumes ofrefolding buffer in a drop-wise manner (about 50 ml refolding bufferadded to about 10 ml of collected elution fraction solution). Therefolding buffer may include about 0.75 M of arginine, about 50 mM ofTris base titrated with hydrochloric acid (Tris-HCl) to a pH of about8.0, about 50 mM of potassium chloride (KCl), and about 0.1 mM of ametal chelator, such as EDTA. The solution was stirred at 4° C. for 48hours.

[0199] After refolding, the recombinant fusion proteins were dialyzed inabout 2 liters of dialysis buffer at about 4° C. for about 24 hours inorder to concentrate the purified recombinant protein for long-termstorage in its inactive form. The dialysis buffer may include about 50mM of Tris-HCl (at a pH of about 7.9), about 50 mM of KCl, about 0.1 mMof EDTA, and about 50% of glycerol. The dialyzed recombinant fusionprotein sample was assayed for protein concentration using a proteinassay kit (Bio-Rad). For example, a total of 5 mg of recombinant SMTGase fusion protein was obtained from the original 250 mlBL21(DE3)+pET28-SMTG E. coli culture, and was stored at −20° C. Inanother experiment, about 3 mg of recombinant SC TGase fusion proteinwas obtained from the original 250 ml BL21 (DE3)+pET28-SCTG E. coliculture, and was stored at −20° C.

[0200]FIG. 4 demonstrated purification of the recombinant SM TGasefusion protein, showing the Coomassie Blue stained SDS-PAGE gel. Lane Mof FIG. 4 is loaded with a mixture of molecular weight markers (fromabout 25 kDa to about 250 Kda). Lane 1 is loaded with cell lysate fromBL21(DE3)+pET28-SMTG E. coli culture without IPTG induction. Lane 2 isloaded with cell lysate from BL21(DE3)+pET28-SMTG E. coli culture withabout 1 mM of IPTG induction. Lane 3 is loaded with the purified6×His-tagged SM TGase fusion protein after the single columnpurification through the His-Bind Ni+column. The purified recombinant SMTGase fusion protein runs at an estimated molecular weight of about 37kDa on the Coomassie Blue stained SDS-PAGE gel. The expression ofrecombiant SM TGase fusion protein was so tightly regulated that onlyafter IPTG induction, the fusion protein was able to be expressed, asindicated by an arrow (present only in lane 2 and 3, but not in lane 1).

[0201] Over-expression and purification of the recombinant SC TGasefusion protein had also been confirmed by performing a Coomassie Bluestained SDS-PAGE gel. Cell lysate from BL21 (DE3)+pET28-SCTG E. coliculture without IPTG induction and cell lysate from BL21(DE3)+pET28-SCTGE. coli culture with about 1 mM of IPTG induction were compared to showover-expression of the recombinant SC TGase fusion protein only whenexpression was induced. The purified 6×His-tagged SC TGase fusionprotein after the single column purification through His-Bind Ni+ columnmigrated as a simple band to an estimated molecular weight of about 40kDa on the Coomassie Blue stained SDS-PAGE gel.

Example 8 Regeneration of Enzymatic Activity of the Purified RecombinantTransqlutaminases

[0202] Cross-linking reactions were set up to assay for cross-linkingactivity of the purified, inactive recombinant SM TGase fusion proteinon a substrate, β-casein. The reactions were incubated at about 25° C.for 16 hours. In some cases, the reactions were incubated at about 37°C. for about 30 minutes or longer, such as about 1 hour or longer. Eachreaction included about 0.005 unit of recombinant transglutaminase per 1mg of 8-casein substrate added to a cross-linking (CL) buffer. The CLbuffer includes about 50 mM of Tris-HCl (at a pH of about 7.4), about20% of glycerol, and various concentration of a reducing agent, DTT.

[0203]FIG. 5 demonstrates the results of exemplary cross-linkingreactions using different reducing agent concentrations. Variousreaction mixtures were loaded to a SDS-PAGE gel and stained withCoomassie Blue to visualize cross-linking of the β-casein substrate. InFIG. 5, lane M represents a mixture of molecular weight markers (fromabout 25 kDa to about 175 kDa). Lane 1 to lane 6 of FIG. 5 represent thecross-linking reaction mixtures in the presence of about 0 mM (lane 1and 2), 2 mM (lane 3 and 4), or 10 mM (lane 5 and 6), of DTT.

[0204] As shown in FIG. 5, in the absence of DTT (lane 1 and 2), therecombinant SM TGase had no cross-linking activity on β-casein andβ-casein runs as a monomer at an estimated molecular weight of 30 kDa.In the presence of CL buffer and about 2 mM of DTT (lane 3 and 4), therecombinant SM TGase was activated and β-casein run as a mixture ofcross-linked polymer and monomer. In the presence of CL buffer and about10 mM of DTT (lane 5 and 6), the recombinant SM TGase was very activeand the catalytic cross-linking activity of SM TGase resulted incross-linking of all detectable 8-casein substrates in the reaction, asjudged by the appearance of a high molecular weight polymer in thestacking gel and the dissapearance of the 30 kDa protein monomers.

[0205] In addition, the concentration of DTT in the CL buffer andactivity of the recombinant SM TGase corresponded to a change of colorof the reaction from clear to yellow. The results are shown in Table 3.The density of the yellow color was measured as absorbance value atOD₄₅₀. As shown in Table 3, there was an increase in OD₄₅₀ value for thereaction mixtures in the present of increased amount of DTT, whichcorrelated to the enzymatic activity of the recombinant SM TGase fusionproteins as shown in FIG. 5. TABLE 3 SM TGase activity correlates withchange of solution color and reducing agent concentration. DTTconcentration 0 mM 2 mM 10 mM OD 450 0.081 0.349 2.012

[0206] The effect of DTT and the change of solution color are unexpectedfeatures of the inactive/active transformation of the exemplaryrecombinant SM TGase and SC TGase fusion proteins, and have not beenobserved previously. The purified recombinant transglutaminases wereassayed on a number of proteins including known substrates andnon-substrates for native microbial transglutaminase.

Example 9 Cross-Linking Proteins by Purified RecombinantTransglutaminases

[0207] An assay was performed to test the cross-linking of β-casein, aknown substrate of native transglutaminases, using the purifiedrecombinant transglutaminase described here. β-casein is available fromSigma-Aldrich as a purified protein in its native form.

[0208] About 5 mg of β-casein (Sigma C-6905) was dissolved inphosphate-buffer-saline (PBS) to a concentration of about 10 mg/ml. Thecross-linking reaction contained about 1 mg of the β-casein incubatedwith about 0.005 unit (about 5 μg, depending on the purification yield)of purified recombinant SM TGase fusion protein in a CL buffercontaining about 50 mM of Tris-HCl (pH 7.4) and about 10 mM of DTT forabout 16 hours at about 25° C. The experimental and control reactionswere loaded on a SDS-PAGE gel and stained with Coomassie Blue after geleletrophoresis.

[0209] The results indicated that when a control reaction containingonly non-cross-linked (Non-CL) β-casein was loaded on a SDS-PAGE gel,the Non-CL β-casein migrated as a monomer. However, when thecross-linking reaction mixture was loaded, the cross-linked (CL)β-casein migrated as a smear on the SDS-PAGE gel indicating a mixture ofhigh molecular weight polymers of different length. The control reactioncontaining only small amount of purified recombinant SM TGase withoutthe β-casein substrate, was loaded on the SDS-PAGE gel, no protein bandwas observed. The enzyme used was approximately 5 μg of purifiedrecombinant SM TGase per 1 mg of β-casein and is not visible byCoomassie Blue staining in the CL reaction mixture and SM TGase controlreaction.

Example 10 Cross-Linking of Recombinant Protein Species by PurifiedRecombinant Transqlutaminases

[0210] Protein species that can serve as substrates of nativetransglutaminases but that were purified as recombinant proteins werealso assayed to see if such purified recombinant protein species couldbe cross-linked by the purified recombinant transglutaminases. Thepurification and cross-linking of two examples of these transglutaminasesubstrates, recombinant serum albumin and recombinant cellulase, aredescribed below.

[0211] Recombinant serum albumin was purified as secreted extracellularprotein from yeast Pichia pastoris GS115 (His+Muts) (available fromInvitrogen) containing an expression vector with a cloned serum albumingene insert. Recombinant serum albumin was harvested from the mediumthrough ammonia sulfate precipitation and centrifugated at a speed ofabout 10,000×g for about 10 minutes to pellet the expressed recombinantserum albumin protein. The purified protein pellet was resuspended in 6Mguanidine-HCl (pH 7.9) and dialyzed in about 100 volumes of excess PBSsolution without the addition of a reducing agent, DTT.

[0212] The dialyzed recombinant serum albumin protein was thenconcentrated by Aguacide (available from Calbiochem). The concentratedsoluble fraction (supernatant) of recombinant serum albumin protein wasassayed for protein concentration using a protein assay kit (availablefrom Bio-Rad).

[0213] Cross-linking of recombinant serum albumin using purifiedrecombinant SM TGase was performed and the cross-linking reactionmixture and control reactions were loaded on a SDS-PAGE gel. Thecross-linking reaction mixture contained about 1 mg of recombinant serumalbumin, incubated with about 0.005 Unit of purified recombinant SMTGase in the presence of the cross-linking buffer (about 50 mM ofTris-HCl, pH 7.4, and about 10 mM of DTT) at about 25° C. for about 16hours.

[0214] First, the control reaction containing only the recombinant serumalbumin migrated as a monomer after 12% SDS-PAGE gel electrophoresis andwas not cross-linked (Non-CL) in the absence of SM TGase. However, thecross-linking reaction containing recombinant serum albumin revealed amixture of very high molecular weight polymers of different length,which migrated as a smear in the stacking gel but not into theseparating gel. Lane 3 contained a control reaction with about 5 ng ofpurified recombinant SMTGase in the reaction, not visible by CoomassieBlue staining.

Example 11 Purification and Cross-Linking of Recombinant Cellulase

[0215] A recombinant cellulase protein was cloned and overexpressed foranalysis by cross-linking using the recombinant SM TGase fusion protein.A cellulase gene from Humicola grisea var. thermoides ATCC 16453 wascloned as a NheI-HindIII DNA fragment from genomic DNA of Humicolagrisea var. thermoides ATCC 16453 using the PCR cloning procedure asdescribed above. Two PCR primers were specifically designed for cloningof cellulase gene, SEQ ID No. 13 and SEQ ID No. 14. The cloned cellulasegene was then subcloned into pET28a (Novagen) expression vector forover-expression and purification of the recombinant cellulase proteinfrom an E. coli host, BL21(DE3). Intracellular recombinant cellulaseprotein was purified through a His-Bind Ni⁺ column (Novagen). The columnwas washed and the recombinant cellulase protein was eluted with aelution buffer containing about 0.5 M of imidazole and about 6M ofguanidine-HCl (pH 7.9). The eluted protein was dialyzed in 100 volumesof excess PBS solution without the refolding agent DTT and thenconcentrated for storage at low temperature.

[0216] The results for the cross-linking of recombinant cellulase usingthe purified recombinant SM TGase were checked on an SDS-PAGE gel. Thecross-linking reaction contained about 1 mg of recombinant cellulase,incubated with about 0.005 Unit of purified recombinant SM TGase in thepresence of the CL buffer (about 50 mM of Tris-HCl, pH 7.4, and about 10mM of DTT) at about 25° C. for various time periods. A control reactionhaving only the recombinant cellulase was not cross-linked (Non-CL) inthe absence of SM TGase and migrated as a monomer after 12% SDS-PAGE gelelectrophoresis. The recombinant cellulase was cross-linked into amixture of very high molecular weight polymers of different length overtime, which migrated as a smear into the stacking gel but not into theseparating gel. Another control reaction with about 5 mg of purifiedrecombinant SMTGase in the reaction showed no protein bands because theamount of recombinant SM TGase used in each reaction was not visible byCoomassie Blue staining.

Example 12 Preparations of Non-Substrate Protein Species to beCross-Linked by Purified Recombinant Transglutaminases

[0217] Protein species that typically cannot serve as substrates fornative transglutaminases were reacted with the purified recombinanttransglutaminase fusion proteins. Native microbial transglutaminase cancross-link only a small number of substrate protein species. Humantransglutaminase Factor XIII has an even narrower substrate spectrum.For the most part, it has been shown that bovine serum albumin (BSA),histone protein, glucose oxidase, ovalbumin, and myelin basic protein(MBP) are all poor substrates for native transglutaminase.

[0218] It has been theorized that most proteins, polypeptides, andpeptides are poor substrates for transglutaminase due to a limitednumber of glutamine and lysine residues in these molecules. In addition,even for molecules that do contain glutamine and lysine, sterichindrance due to folding into three-dimensional structures may result inno cross-linking activity.

[0219] When the purified recombinant transglutaminase fusion proteinswere used initially to cross-link non-substrate proteins, such as bovineserum albumin (BSA), histone protein, glucose oxidase, ovalbumin, andmyelin basic protein (MBP) (all available from Sigma), there was nocross-linking even in the presence of a large amount of the purifiedrecombinant transglutaminase fusion proteins in the activation solution.For example, even in the presence of 10 mM DTT in the CL buffer, therewas no cross-linking of the non-substrate native proteins. However, itwas found through experimetation that modification of the proteinsand/or specific preparation of the proteins to be used in thecross-linking reaction resulted in the cross-linking of a broad range ofprotein species by the purified recombinant transglutaminases.

[0220] About 10 mg of each of BSA, histone protein, glucose oxidase, andovalbumin, were denatured in 10 ml of about 6M of guanidine-HCl (pH7.9). After denaturation, each protein sample was dialyzed in 2 litersof PBS solution without the reducing agent, DTT, at about 4° C. forabout 24 hours. It is thought that the denatured proteins are partiallyrefolded after dialysis without the addition of the reducing agent.

[0221] After dialysis, the samples were centrigued at 10,000 xg forabout 10 minutes and minor precipitation was discarded. The solublesupernatant sample was assayed for protein concentration using a proteinassay kit (Bio-Rad) and diluted in PBS solution to about 5 mg/ml. If thesample concentration was less than about 2 mg/ml, the sample wasconcentrated through Aquacide (Calbiochem) in dialysis bags to aconcentration of at least about 2 mg/ml.

Example 13 Cross-Linking of Modified Non-Substrate Protein Species byPurified Recombinant Transglutaminases

[0222] The denatured and partially refolded protein species, such asbovine serum albumin (BSA), histone, glucose oxidase, and ovalbumin, aswell as the native protein species of bovine serum albumin (BSA),histone, glucose oxidase, and ovalbumin were cross-linked in the CLbuffer containing about 50 mM of Tris-HCl (pH 7.4) and about 10 mM ofDTT in the presence of about 0.05 unit of purified recombinant SM TGaseper 1 mg of modified proteins at about 25° C. for about 16 hours. Theresulting reactions were applied to 12% SDS-PAGE and stained withCoomassie Blue after gel electrophoresis. In each experimentcross-linking reactions for both native protein and modified proteinwere prepared.

[0223] The results from SDS-PAGE showed that only the reactionscontaining the modified protein species of BSA, histine H3 protein,glucose oxidase, and ovalbumin were cross-linked by the purifiedrecombinant SM TGase. The cross-linked products of these modifiedproteins migrated as a smear in the stacking gel, indicating theproduction of a mixture of high molecular weight cross-linked polymersby recombinant transglutaminases. Control reactions having only nativeproteins or modified proteins without added purified recombinant SMTGase were also checked on SDS-PAGE. The non-cross-linked proteinsmigrated as a monomer. Note that the SM TGase used was about 10 foldhigher in amount (about 50 μg) and can be stained by Coomassie Blue. Theresults from the cross-linked modified non-substrate protein speciessuggested that modified protein samples (through denaturation anddialysis) are cross-linked to a far greater extent than the nativeprotein samples. Notethat complete and partial cross-linking was bothobserved for native histine H3 protein under the condition tested.

Example 14 Cross-linking of Two or More Protein Species by PurifiedRecombinant Transqlutaminases

[0224] The purified recombinant transglutaminases were used tocross-link a mixture of proteins/polypeptides. For example,cross-linking of a mixture of 8-casein and glucose oxidase by purifiedrecombinant SM TGase was performed, using about 0.005 unit of SM TGaseper 1 mg of combined β-casein and glucose oxidase, and was checked onSDS-PAGE. As another example, cross-linking of cellulase and serumalbumin by purified recombinant SM TGase was also performed, using about0.005 unit of SM TGase per 1 mg of combined cellulase and serum albumin.In both experiments, the cross-linking reactions migrated as a smearpresent in the stacking gel indicating cross-linking of both proteinspecies into a mixture of high molecular weight cross-linked polymers byrecombinant transglutaminases. Control reactions without added purifiedrecombinant SM TGase confirmed the migration of the non-cross-linkedproteins to their respective monomer positions. Control reactions withonly the purified recombinant SM TGase resulted in no protein bandbecause the amount of purified SM TGase used cannot be stained byCoomassie Blue. In each experiment, both protein species can becross-linked by the purified recombinant SM TGase, as indicated by thedisappearance of the two monomer bands.

Example 15 Cross-Linking of Naturally-Occurring Peptides by PurifiedRecombinant Transqlutaminases

[0225] The purified recombinant transglutaminases described herein wereused to cross-link short chains of naturally-occurring or syntheticpeptides that have internal glutamine and lysine residues (not on theN-terminus or C-Terminus). One example is the naturally-occurringβ-amyloid peptide (1-42) which plays an important role during thepathogenesis of Alzheimer's disease.

[0226]FIG. 6 illustrates cross-linking of β-amyloid peptide (1-42, SEQID NO. 15, DAEFRHDSGTEVHHQKLVFFAEDVGSNKGAIIGLMVG GVVIA 42 amino acide bypurified recombinant transglutaminases, using about 0.05 unit (about 50μg) of purified recombinant SM TGase per 1 mg of β-amyloid peptide. Theβ-amyloid (1-42) peptide (purchased from American Peptide Company(Sunnyvale, Calif.) includes 1 glutamine (Gln, Q) residue and 2 lysine(Lys, K) residues that can be reacted with transglutaminase.

[0227] In FIG. 6, lanes 2-4 contained the cross-linking reactionsincubated for about 1 hour, 2 hours and 3 hours, respectively. Theβ-amyloid (1-42) peptide can be cross-linked by the recombinanttransglutaminase as shown as a smear of a mixture of cross-linkedpeptides on the top of the separating gel and the disappearance of thepeptide bands at the bottom of the SDS-PAGE. Lane 1 of FIG. 6 containedthe control reaction β-amyloid peptide only) without added purifiedrecombinant SM TGase, showing the migration of the non-cross-linkedpeptides at the bottom of the SDS-PAGE.

Example 16 Cross-Linking of Synthetic Peptides by Purified RecombinantTransqlutaminases

[0228] The purified recombinant transglutaminases were also used tocross-link short chain synthetic peptides that have glutamine and lysineresidues on the N-terminus or C-terminus. Clearly, if there are noglutamine and/or lysine residues in the amino acid sequence of theproteins, polypeptides, and peptides to be cross-linked bytransglutaminase, the proteins, polypeptides, and peptides can bemodified to include glutamine and/or lysine residues on the N-terminusor C-terminus.

[0229] For example, cross-linking activity of the purified recombinanttransglutaminase was assayed on a peptide, BSA5. The peptide sequence ofBSA5 (SEQ ID No.16, KKKCCTESLVNRRPCFSQQQ, 20 amino acids) was designedand synthesized from ResGen (Invitrogen) to include with 3 extra lysineresidues on the amino (N) terminus and 3 extra glutamine residues on thecarboxyl (C) terminus. The peptide sequence of BSA5 also includes anamino acid sequence of about 14 amino acids (residue number 4 to 17 inBSA5 peptide), according to the sequence of BSA protein from Bos taurusand corresponding to part of C-terminal conserved portion of bovineserum albumin (BSA) protein family.

[0230] BSA5 peptide was synthesized and stored in PBS solution to becross-linked with SM TGase. The cross-linking reaction was set up usingabout 0.05 unit (about 50 μg) of SM TGase per 1 mg of BSA5 peptide in CLbuffer at about 25° C. for about 16 hours.

[0231]FIG. 7 is a Coomassie Blue stained SDS-PAGE gel and illustratescross-linking of BSA5 peptide by purified recombinant transglutaminasefusion protein. Lane 1 contained non-cross-linked BSA 5 peptide monomer.Lanes 2-5 contained the cross-linking reaction in the presence ofincreased amount of recombinant SM TGase corresponding to an increase incross-linked (CL) BSA5 peptide, which run as a large molecular weightpolymer on the top of the separating gel. Lane 6 contained only purifiedrecombinant SM TGase (about 50 ng) as a control.

[0232] The results of the cross-linking experiments described abovesuggest that the purified recombinant transglutaminases exhibit highenzymatic activity on a variety of substrates, modified non-substrates,and mixtures of two or more substrates, including proteins,polypeptides, peptides, to generate different lengths of cross-linkedhomo-polymers and even hetero-polymers. The method of cross-linkingusing transglutaminases and the cross linked products provide a powerfultool to be used in many applications, including but not limited to,vaccine development and immunotherapy.

Example 17 Immunization with Cross-Linked Products

[0233] Cross-linked products such as those examples described above wereused as antigens to induce a high level of antibody production in mouse,as compared to the lower level of antibody production in response to theuse of non-cross-linked monomer antigens. For the experiments describedbelow, about 100 μg of non-cross-linked proteins, polypeptides, andpeptides, and cross-linked products were diluted in about 0.5 ml PBSsolution for use in immunizing a mouse (Southwest mouse, 8-12 weeksold). In addition, about 0.8 mg of aluminum hydroxide was used asadjuvant.

[0234] For example, four mice were immunized with about 100 μg ofβ-casein and four mice were immunized with about 100 μg of cross-linkedβ-casein. The mice were immunized at day 1 and day 21, and serum wascollected at day 28 from each mouse. Collected sera were titered byenzyme-linked immunosorbent assay (ELISA) using the following procedure.An ELISA plate was coated with 1 μg/100 μl/well of β-casein (Non-CL) inPBS solution at about 4° C. for about 16 hours. After coating, the platewas washed three times with 200 μl/well of wash buffer containing 1× PBSplus 0.05% Tween 20. After washing, the plate was blocked in blockingbuffer containing 1% bovine serum albumin (BSA) in wash buffer at roomtemperature for 1 hour. The plate was washed three times with washbuffer of about 200 μl/well. Sera collected from day 28 immunized micewere serially diluted half from 1:100 to 1:409600 in blocking buffer.Series of diluted sera were loaded onto the ELISA plate at about 100μl/well in duplicate wells and incubated at room temperature for about 1hour. The plate was washed three times with wash buffer of about 200μl/well. Peroxidase conjugated anti-mouse secondary antibody was dilutedin blocking buffer (1: 2500) and loaded onto the ELISA plate at 100μl/well and incubated at room temperature for about 1 hour. The platewas washed three times with wash buffer and then developed using about100 μl/well of peroxidase substrate. The plate was incubated at roomtemperature for 30 minutes, and the color developed was stopped withabout 4N of hydrogen sulfate (H₂SO₄).

[0235] The color-developed ELISA plate was pictured and measured inELISA reader at 450 nm absorbance The duplicated value was averaged andthe results were plotted into FIGS. 8-11 and discussed below (anti-seracollected from four mice of the same immunogen injection were averagedand standard deviation for each set of experiment was shown).

Example 18 Immunization Using Cross-Linked Products of Native Proteinsas Antigens

[0236] ELISA results for the anti-sera obtained from mice immunized withcross-linked and non-cross-linked native β-casein protein were obtained.The results suggest that cross-linked native proteins can be used toinduce antibody production and the anti-sera obtained from thecross-linked β-casein react or bind much stronger to β-casein than theanti-sera obtained from the non-cross-linked β-casein. The resultingOD₄₅₀ value from the ELISA assay is shown in Table 4 and is about 10:1or more for cross-linked versus non-cross-linked. More importantly, thetiter of the anti-sera of the cross-linked β-casein is much higher thanthat of the non-cross-linked β-casein. Significantly, the titer of theanti-sera is calculated to be increased to about 128 fold or more(cross-linked versus non-cross-linked). TABLE 4 450 nm absorbance valuesfor various anti-sera from mice immunized with cross-linked andnon-cross-linked native β-casein protein as assayed on ELISA platecoated with β-casein Serum dilution Mice 1:100 1:200 1:400 1:800 1:16001:3200 Non-CL 1 0.954 0.558 0.332 0.223 0.184 0.165 Non-CL 2 1.121 .0673.0371 0.245 0.163 0.161 Non-CL 3 0.894 0.509 .0315 0.194 0.142 0.150Non-CL 4 0.781 .0432 0.239 0.159 0.144 0.134 CL-1 2.153 2.122 2.1112.101 1.900 1.623 CL-2 2.159 2.115 2.034 1.970 1.744 1.430 CL-3 2.0982.123 2.020 1.926 1.755 1.427 CL-4 2.093 2.088 1.996 1.867 1.625 1.225Serum dilution Mice 1:6400 1:12800 1:25600 1:51200 1:102400 Blank Non-CL1 0.087 0.082 0.082 0.084 0.084 0.082 Non-CL 2 0.086 0.083 0.084 0.0820.079 0.081 Non-CL 3 0.087 0.081 0.081 0.078 0.084 0.079 Non-CL 4 0.0790.081 0.078 0.079 0.080 0.080 CL-1 1.237 0.795 0.432 0.226 0.158 0.081CL-2 0.939 0.484 0.237 0.169 0.113 0.080 CL-3 0.996 0.537 0.300 0.1810.122 0.083 CL-4 0.805 0.420 0.232 0.145 0.114 0.082

Example 19 Immunization Using Cross-Linked Products of RecombinantProteins as Antigens

[0237] Cross-linked recombinant proteins, such as cross-linkedrecombinant serum albumin and cross-linked recombinant cellulase werealso used as antigens to immunize mice. ELISA results for the anti-seraobtained from mice immunized with the cross-linked and non-cross-linkedrecombinant serum albumin were obtained. The results suggest that theanti-sera obtained from the cross-linked recombinant serum albumin willreact or bind much stronger to recombinant serum albumin than theanti-sera obtained from the non-cross-linked recombinant serum albumin.The resulting OD₄₅₀ value is shown in Table 5 and is about 10:1 or morefor cross-linked versus non-cross-linked recombinant serum albumin.Significantly, the titer of the anti-sera is calculated to be increasedto about 64 fold or more (cross-linked versus non-cross-linked). TABLE 5450 nm absorbance values for various anti-sera from mice immunized withcross-linked and non-cross-linked recombinant serum albumin as assayedon ELISA plate coated with cellulase Serum dilution Mice 1:100 1:2001:400 1:800 1:1600 1:3200 Non-CL 1 0.881 0.562 0.310 0.224 0.168 0.141Non-CL 2 0.768 0.443 0.301 0.178 0.137 0.121 Non-CL 3 0.824 0.512 0.2980.201 0.132 0.125 Non-CL 4 0.781 0.412 0.224 0.154 0.124 0.118 CL-12.110 2.079 2.043 1.935 1.698 1.425 CL-2 2.088 2.071 2.014 1.923 1.7031.415 CL-3 2.015 2.011 1.985 1.779 1.535 1.247 CL-4 2.210 2.105 2.0361.956 1.702 1.433 Serum dilution Mice 1:6400 1:12800 1:25600 1:512001:102400 Blank Non-CL 1 0.084 0.081 0.080 0.084 0.087 0.088 Non-CL 20.091 0.071 0.084 0.079 0.083 0.089 Non-CL 3 0.082 0.078 0.088 0.0750.085 0.079 Non-CL 4 0.082 0.077 0.086 0.073 0.084 0.078 CL-1 0.8890.516 0.310 0.173 0.118 0.081 CL-2 0.902 0.479 0.274 0.149 0.105 0.085CL-3 0.769 0.385 0.2210 0.122 0.092 0.082 CL-4 0.921 0.526 0.342 0.1820.121 0.077

[0238] ELISA results for the anti-sera obtained from mice immunized withthe cross-linked and non-cross-linked recombinant cellulase were alsoobtained. The results suggest that the anti-sera obtained from thecross-linked recombinant cellulase will react or bind much stronger torecombinant serum cellulase the anti-sera obtained from thenon-cross-linked recombinant cellulose. The resulting OD₄₅₀ value isshown in Table 6 and is about 8:1 or more for cross-linked versusnon-cross-linked recombinant cellulose. Significantly, the titer of theanti-sera is calculated to be increased to about 20 fold or more(cross-linked versus non-cross-linked). TABLE 6 450 nm absorbance valuesfor various anti-sera from mice immunized with cross-linked andnon-cross-linked recombinant cellulase as assayed on ELISA plate coatedwith cellulase Serum dilution Mice 1:100 1:200 1:400 1:800 1:1600 1:3200Non-CL 1 0.621 0.302 0.146 0.112 0.089 0.082 Non-CL 2 0.714 0.401 0.2110.162 0.138 0.129 Non-CL 3 0.771 0.432 0.253 0.172 0.141 0.123 Non-CL40.811 0.481 0.289 0.201 0.151 0.141 CL-1 1.975 1.742 1.593 1.324 1.0150.784 CL-2 2.012 1.824 1.496 1.215 0.964 0.622 CL-3 1.894 1.642 1.3561.112 0.812 0.405 CL-4 1.912 1.688 1.412 1.135 0.902 0.521 Serumdilution Mice 1:6400 1:12800 1:25600 1:51200 1:102400 Blank Non-CL 10.085 0.076 0.086 0.075 0.087 0.078 Non-CL 2 0.085 0.071 0.084 0.0800.076 0.081 Non-CL 3 0.078 0.085 0.084 0.079 0.072 0.074 Non-CL 4 0.0960.084 0.076 0.081 0.077 0.084 CL-1 0.301 0.256 0.166 0.094 0.082 0.081CL-2 0.288 0.179 0.095 0.081 0.079 0.082 CL-3 0.197 0.102 0.084 0.0780.075 0.077 CL-4 0.259 0.148 0.089 0.083 0.081 0.085

Example 20 Immunization Using Cross-Linked Products of ModifiedNon-Substrate Proteins as Antigens

[0239] Cross-linked modified non-substrate proteins, such ascross-linked glucose oxidase, were also used as antigens to immunizemice. ELISA results for the anti-sera obtained from mice immunized withmodified non-substrate proteins antigens, the cross-linked andnon-cross-linked glucose oxidase, were obtained. The results suggestthat the anti-sera obtained from the cross-linked glucose oxidase willreact or bind much stronger to glucose oxidase than the anti-seraobtained from the non-cross-linked glucose oxidase (OD₄₅₀ value wasabout 12:1 or more for cross-linked versus non-cross-linked as shown inTable 7). Significantly, the titer of the anti-sera is calculated to beincreased to about 64 fold or more (cross-linked versusnon-cross-linked). TABLE 7 450 nm absorbance values for variousanti-sera from mice immunized with cross-linked and non-cross-linkedmodified glucose oxidase as assayed on ELISA plate coated with glucoseoxidase Serum dilution Mice 1:100 1:200 1:400 1:800 1:1600 1:3200 Non-CL1 0.785 0.421 0.241 0.162 0.141 0.091 Non-CL 2 0.912 0.524 0.321 0.2420.153 0.137 Non-CL 3 0.776 0.425 0.223 0.161 0.145 0.125 Non-CL 4 0.8890.511 0.302 0.224 0.149 0.132 CL-1 2.252 2.241 2.125 2.013 1.921 1.598CL-2 2.158 2.115 2.052 2.021 1.812 1.457 CL-3 2.198 2.101 1.986 1.8211.752 1.321 CL-4 2.168 2.116 2.032 1.925 1.798 1.465 Serum dilution Mice1:6400 1:12800 1:25600 1:51200 1:102400 Blank Non-CL 1 0.087 0.088 0.0910.079 0.081 0.081 Non-CL 2 0.086 0.091 0.087 0.082 0.086 0.079 Non-CL 30.088 0.081 0.078 0.089 0.079 0.082 Non-CL 4 0.092 0.086 0.085 0.0820.087 0.085 CL-1 1.241 0.779 0.354 0.21 0.145 0.086 CL-2 1.121 0.6970.325 0.195 0.143 0.078 CL-3 0.987 0.596 0.258 0.168 0.119 0.087 CL-41.028 0.621 0.305 0.175 0.126 0.085

Example 21 Immunization Using Cross-Linked Products of Protein Mixturesas Antigens

[0240] Cross-linked protein mixtures, such as cross-linking products ofa mixture of two or more proteins, were also used as antigens toimmunize mice. ELISA results for the anti-sera obtained from miceimmunized using cross-linked and non-cross-linked protein mixtures asantigens were obtained. For example, anti-sera against cross-linkedproducts of protein mixture containing β-casein and glucose oxidase(about 200 μg of total protein) were obtained. The anti-sera obtainedwere assayed on ELISA plate coated with either β-casein or glucoseoxidase and the results were plotted. The anti-sera obtained from thecross-linked mixtures reacted or bound much more strongly to β-caseinthan the anti-sera obtained from the non-cross-linked mixtures. Theresulting OD₄₅₀ value is about 10:1 or more for cross-linked versusnon-cross-linked as shown in Table 8. Significantly, the titer of theanti-sera is calculated to be increased to about 64 fold or more(cross-linked versus non-cross-linked). TABLE 8 450 nm absorbance valuesfor various anti-sera from mice immunized with cross-linked andnon-cross-linked β-casein and glucose oxidase mixtures as assayed onELISA plate coated with β-casein Serum dilution Mice 1:100 1:200 1:4001:800 1:1600 1:3200 Non-CL 1 1.015 0.621 0.321 0.232 0.159 0.138 Non-CL2 1.211 0.691 0.381 0.263 0.192 0.175 Non-CL 3 0.846 0.472 0.281 0.1920.126 0.088 Non-CL 4 0.951 0.569 0.302 0.211 0.165 0.121 CL-1 2.0882.021 1.892 1.745 1.378 1.026 CL-2 2.114 2.031 2.002 1.865 1.724 1.428CL-3 2.121 2.113 2.067 1.987 1.801 1.521 CL-4 2.085 2.005 1.881 1.7981.354 1.102 Serum dilution Mice 1:6400 1:12800 1:25600 1:51200 1:102400Blank Non-CL 1 0.078 0.082 0.085 0.075 0.084 0.086 Non-CL 2 0.095 0.0860.081 0.086 0.080 0.084 Non-CL 3 0.075 0.084 0.083 0.078 0.081 0.075Non-CL 4 0.082 0.088 0.084 0.078 0.077 0.084 CL-1 0.785 0.511 0.2810.124 0.095 0.078 CL-2 0.921 0.668 0.341 0.176 0.118 0.080 CL-3 1.0470.761 0.403 0.217 0.170 0.081 CL-4 0.742 0.477 0.259 0.106 0.087 0.089

[0241] Advantageously, in another ELISA experiment, the same anti-seraalso react to glucose oxidate. The resulting OD₄₅₀ value is about 8:1 ormore for cross-linked versus non-cross-linked as shown in Table 9 andthe titer of the anti-sera is thus calculated to be increased to about32 fold or more for the cross-linked mixtures versus non-cross linkedmixtures. TABLE 9 450 nm absorbance values for various anti-sera frommice immunized with cross-linked and non-cross-linked β-casein andglucose oxidase mixtures as assayed on ELISA plate coated with glucoseoxidase Serum dilution Mice 1:100 1:200 1:400 1:800 1:1600 1:3200 Non-CL1 0.672 0.325 0.221 0.143 0.125 0.085 Non-CL 2 0.841 0.441 0.277 0.1710.136 0.091 Non-CL 3 0.669 0.345 0.237 0.142 0.129 0.089 Non-CL 4 0.6910.367 0.242 0.151 0.130 0.084 CL-1 2.015 1.997 1.826 1.546 1.136 0.788CL-2 1.985 1.846 1.528 1.102 0.779 0.441 CL-3 2.221 2.106 1.895 1.7421.358 1.108 CL-4 1.962 1.823 1.486 1.089 0.748 0.389 Serum dilution Mice1:6400 1:12800 1:25600 1:51200 1:102400 Blank Non-CL 1 0.084 0.083 0.0780.077 0.074 0.082 Non-CL 2 0.087 0.076 0.088 0.084 0.072 0.079 Non-CL 30.085 0.081 0.073 0.082 0.083 0.077 Non-CL 4 0.075 0.082 0.081 0.0850.078 0.083 CL-1 0.421 0.258 0.132 0.089 0.085 0.075 CL-2 0.236 0.1680.091 0.079 0.082 0.083 CL-3 0.756 0.448 0.216 0.181 0.095 0.085 CL-40.221 0.129 0.085 0.082 0.078 0.073

[0242]FIGS. 8 and 9 showed the ELISA results of cross-linked products ofprotein mixtures containing serum albumin and cellulase (about 200 μg oftotal protein). The anti-sera obtained were assayed on ELISA platecoated with either serum albumin or cellulase and the results wereplotted in FIGS. 8 and 9, respectively. In FIG. 8, the anti-seraobtained from the cross-linked mixtures reacted or bound much morestrongly to serum albumin than the anti-sera obtained from thenon-cross-linked mixtures. The resulting OD₄₅₀ value was about 7:1 ormore for cross-linked protein mixture versus non-cross-linked proteinmixture. Significantly, the titer of the anti-sera is calculated to beincreased to about 32 fold or more (cross-linked versusnon-cross-linked). Advantageously, in FIG. 9, the same anti-sera alsoreact to cellulose. The resulting OD₄₅₀ value is about 8:1 or more forcross-linked versus non-cross-linked and the titer of the anti-sera iscalculated to be increased to about 32 fold or more for the cross-linkedmixtures versus non-cross-linked mixtures.

Example 22 Immunization Using Cross-Linked Products of Peptides asAntigens

[0243] Cross-linked peptides, such as cross-linked β-amyloid peptide andcross-linked BSA5 peptide, were also used as antigens to immunize miceand the results are shown in FIGS. 10-11.

[0244]FIG. 10 illustrates the ELISA results for the anti-sera obtainedfrom mice immunized with peptide antigens, the cross-linked andnon-cross-linked β-amyloid peptide. The results suggest that theanti-sera obtained from the cross-linked β-amyloid peptide reacted orbound much more strongly to β-amyloid peptide than the anti-seraobtained from the non-cross-linked β-amyloid peptide (OD₄₅₀ value wasabout 6:1 or more for cross-linked versus non-cross-linked).Significantly, the titer of the anti-sera is calculated to be increasedto about 10 fold or more (cross-linked versus non-cross-linked).

[0245]FIG. 11 illustrates the ELISA results for the anti-sera obtainedfrom mice immunized with peptide antigens, the cross-linked andnon-cross-linked BSA5 peptide. The results suggest that the anti-seraobtained from the cross-linked BSA5 peptide reacted or boud much morestrongly to BSA5 peptide than the anti-sera obtained from thenon-cross-linked BSA5 peptide (OD₄₅₀ value is about 7:1 or more forcross-linked versus non-cross-linked). Significantly, the titer of theanti-sera is calculated to be increased to about 32 fold or more(cross-linked versus non-cross-linked).

[0246] While the foregoing is directed to embodiments of the invention,other and further embodiments of the invention may be devised withoutdeparting from the basic scope thereof, and the scope thereof isdetermined by the claims that follow.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 16 <210> SEQ ID NO 1<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Streptomycesmobaraensis ATCC 29032 <400> SEQUENCE: 1 gctagccccg actccgacga cagggtc27 <210> SEQ ID NO 2 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:Streptomyces mobaraensis ATCC 29032 <400> SEQUENCE: 2 tcacggccagccctgcttta ccttg 25 <210> SEQ ID NO 3 <211> LENGTH: 28 <212> TYPE: DNA<213> ORGANISM: Streptomyces cinnamoneus ATCC 11874 <400> SEQUENCE: 3gctagctccc gggccccctc cgatgacc 28 <210> SEQ ID NO 4 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Streptomyces cinnamoneus ATCC 11874<400> SEQUENCE: 4 tcacggccag ccttgctcca ccttgg 26 <210> SEQ ID NO 5<211> LENGTH: 998 <212> TYPE: DNA <213> ORGANISM: Streptomycesmobaraensis ATCC 29032 <400> SEQUENCE: 5 cccgactccg acgacagggtcacccctccc gccgagccgc tcgacaggat gcccgacccg 60 taccgtccct cgtacggcagggccgagacg gtcgtcaaca actacatacg caagtggcag 120 caggtctaca gccaccgcgacggcaggaag cagcagatga ccgaggagca gcgggagtgg 180 ctgtcctacg gctgcgtcggtgtcacctgg gtcaattcgg gtcagtaccc gacgaacaga 240 ctggccttcg cgtccttcgacgaggacagg ttcaagaacg agctgaagaa cggcaggccc 300 cggtccggcg agacgcgggcggagttcgag ggccgcgtcg cgaaggagag cttcgacgag 360 gagaagggct tccagcgggcgcgtgaggtg gcgtccgtca tgaacagggc cctggagaac 420 gcccacgacg agagcgcttacctcgacaac ctcaagaagg aactggcgaa cggcaacgac 480 gccctgcgca acgaggacgcccgttccccg ttctactcgg cgctgcggaa cacgccgtcc 540 ttcaaggagc ggaacggaggcaatcacgac ccgtccagga tgaaggccgt catctactcg 600 aagcacttct ggagcggccaggaccggtcg agttcggccg acaagaggaa gtacggcgac 660 ccggacgcct tccgccccgccccgggcacc ggcctggtcg acatgtcgag ggacaggaac 720 attccgcgca gccccaccagccccggtgag ggattcgtca atttcgacta cggctggttc 780 ggcgcccaga cggaagcggacgccgacaag accgtctgga cccacggaat cactatcacg 840 cgcccaatgg cagcctgggtgccatgcatg tctacgagag caagttccgc aactggtccg 900 agggttactc ggacttcgaccgcggagcct atgtgatcac cttcatcccc aagagctgga 960 acaccgcccc cgacaaggtaaagcagggct ggccgtga 998 <210> SEQ ID NO 6 <211> LENGTH: 332 <212> TYPE:PRT <213> ORGANISM: Streptomyces mobaraensis ATCC 29032 <400> SEQUENCE:6 Pro Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg 1 5 1015 Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val 20 2530 Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly 35 4045 Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly 50 5560 Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg 65 7075 80 Leu Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys 8590 95 Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg100 105 110 Val Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg AlaArg 115 120 125 Glu Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala HisAsp Glu 130 135 140 Ser Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala AsnGly Asn Asp 145 150 155 160 Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro PheTyr Ser Ala Leu Arg 165 170 175 Asn Thr Pro Ser Phe Lys Glu Arg Asn GlyGly Asn His Asp Pro Ser 180 185 190 Arg Met Lys Ala Val Ile Tyr Ser LysHis Phe Trp Ser Gly Gln Asp 195 200 205 Arg Ser Ser Ser Ala Asp Lys ArgLys Tyr Gly Asp Pro Asp Ala Phe 210 215 220 Arg Pro Ala Pro Gly Thr GlyLeu Val Asp Met Ser Arg Asp Arg Asn 225 230 235 240 Ile Pro Arg Ser ProThr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp 245 250 255 Tyr Gly Trp PheGly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val 260 265 270 Trp Thr HisGly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala 275 280 285 Met HisVal Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser 290 295 300 AspPhe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp 305 310 315320 Asn Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 325 330 <210> SEQ IDNO 7 <211> LENGTH: 1005 <212> TYPE: DNA <213> ORGANISM: Streptomycescinnamoneus ATCC 11874 <400> SEQUENCE: 7 tcccgggccc cctccgatgaccgggaaact cctcccgccg agccgctcga caggatgcct 60 gaggcgtacc gggcctacggaggcagggcc actacggtcg tcaacaacta catacgcaag 120 tggcagcagg tctacagtcaccgcgacgga aagaaacagc aaatgaccga agagcagcga 180 gaaaagctgt cctacggttgcgttggcgtc acctgggtca actcgggccc ctacccgacg 240 aacagattgg cgttcgcgtccttcgacgag aacaagtaca agaacgacct gaagaacacc 300 agcccccgac ccgatgaaacgcgggcggag ttcgagggtc gcatcgccaa gggcagtttc 360 gacgagggga agggtttcaagcgggcgcgt gatgtggcgt ccgtcatgaa caaggccctg 420 gaaaatgccc acgacgaggggacttacatc aacaacctca agacggagct cacgaacaac 480 aatgacgctc tgctccgcgaggacagccgc tcgaacttct actcggcgct gaggaacaca 540 ccgtccttca aggaaagggacggcggcaac tacgacccgt ccaagatgaa ggcggtgatc 600 tactcgaagc acttctggagcgggcaggac cagcggggct cctccgacaa gaggaagtac 660 ggcgacccgg aagccttccgccccgaccag ggtaccggcc tggtcgacat gtcgaaggac 720 agaagcattc cgcgcagtccggccaagccc ggcgaaggtt gggtcaattt cgactacggt 780 tggttcgggg ctcaaacagaagcggatgcc gacaaaacca catggaccca cggcgaccac 840 taccacgcgc ccaatagcgacctgggcccc atgcacgtac acgagagcaa gttccggaag 900 tggtctgccg ggtacgcggacttcgaccgc ggagcctacg tgatcacgtt catacccaag 960 agctggaaca ccgcccccgccaaggtggag caaggctggc cgtga 1005 <210> SEQ ID NO 8 <211> LENGTH: 335<212> TYPE: PRT <213> ORGANISM: Streptomyces cinnamoneus ATCC 11874<400> SEQUENCE: 8 Ser Arg Ala Pro Ser Asp Asp Arg Glu Thr Pro Pro AlaGlu Pro Leu 1 5 10 15 Asp Arg Met Pro Glu Ala Tyr Arg Ala Tyr Gly GlyArg Ala Thr Thr 20 25 30 Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln ValTyr Ser His Arg 35 40 45 Asp Gly Lys Lys Gln Gln Met Thr Glu Glu Gln ArgGlu Lys Leu Ser 50 55 60 Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser GlyPro Tyr Pro Thr 65 70 75 80 Asn Arg Leu Ala Phe Ala Ser Phe Asp Glu AsnLys Tyr Lys Asn Asp 85 90 95 Leu Lys Asn Thr Ser Pro Arg Pro Asp Glu ThrArg Ala Glu Phe Glu 100 105 110 Gly Arg Ile Ala Lys Gly Ser Phe Asp GluGly Lys Gly Phe Lys Arg 115 120 125 Ala Arg Asp Val Ala Ser Val Met AsnLys Ala Arg Leu Glu Asn Ala 130 135 140 His Asp Glu Gly Thr Tyr Ile AsnAsn Leu Lys Thr Glu Leu Thr Asn 145 150 155 160 Asn Asn Asp Ala Leu LeuArg Glu Asp Ser Arg Ser Asn Phe Tyr Ser 165 170 175 Ala Leu Arg Asn ThrPro Ser Phe Lys Glu Arg Asp Gly Gly Asn Tyr 180 185 190 Asp Pro Ser LysMet Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser 195 200 205 Gly Gln AspGln Arg Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro 210 215 220 Glu AlaPhe Arg Pro Asp Gln Gly Thr Gly Leu Val Asp Met Ser Lys 225 230 235 240Asp Arg Ser Ile Pro Arg Ser Pro Ala Lys Pro Gly Glu Gly Trp Val 245 250255 Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp 260265 270 Lys Thr Thr Trp Thr His Gly Asp His Tyr His Ala Pro Asn Ser Asp275 280 285 Leu Gly Pro Met His Val His Glu Ser Lys Phe Arg Lys Trp SerAla 290 295 300 Gly Tyr Ala Asp Phe Asp Arg Gly Ala Tyr Val Ile Thr PheIle Pro 305 310 315 320 Lys Ser Trp Asn Thr Ala Pro Ala Lys Val Glu GlnGly Trp Pro 325 330 335 <210> SEQ ID NO 9 <211> LENGTH: 1067 <212> TYPE:DNA <213> ORGANISM: Streptomyces mobaraensis ATCC 29032 <400> SEQUENCE:9 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggctagcc ccgactccga cgacagggtc acccctcccg ccgagccgct cgacaggatg 120cccgacccgt accgtccctc gtacggcagg gccgagacgg tcgtcaacaa ctacatacgc 180aagtggcagc aggtctacag ccaccgcgac ggcaggaagc agcagatgac cgaggagcag 240cgggagtggc tgtcctacgg ctgcgtcggt gtcacctggg tcaattcggg tcagtacccg 300acgaacagac tggccttcgc gtccttcgac gaggacaggt tcaagaacga gctgaagaac 360ggcaggcccc ggtccggcga gacgcgggcg gagttcgagg gccgcgtcgc gaaggagagc 420ttcgacgagg agaagggctt ccagcgggcg cgtgaggtgg cgtccgtcat gaacagggcc 480ctggagaacg cccacgacga gagcgcttac ctcgacaacc tcaagaagga actggcgaac 540ggcaacgacg ccctgcgcaa cgaggacgcc cgttccccgt tctactcggc gctgcggaac 600acgccgtcct tcaaggagcg gaacggaggc aatcacgacc cgtccaggat gaaggccgtc 660atctactcga agcacttctg gagcggccag gaccggtcga gttcggccga caagaggaag 720tacggcgacc cggacgcctt ccgccccgcc ccgggcaccg gcctggtcga catgtcgagg 780gacaggaaca ttccgcgcag ccccaccagc cccggtgagg gattcgtcaa tttcgactac 840ggctggttcg gcgcccagac ggaagcggac gccgacaaga ccgtctggac ccacggaatc 900actatcacgc gcccaatggc agcctgggtg ccatgcatgt ctacgagagc aagttccgca 960actggtccga gggttactcg gacttcgacc gcggagccta tgtgatcacc ttcatcccca 1020agagctggaa caccgccccc gacaaggtaa agcagggctg gccgtga 1067 <210> SEQ ID NO10 <211> LENGTH: 355 <212> TYPE: PRT <213> ORGANISM: Streptomycesmobaraensis ATCC 29032 (about 355 amino acids) <400> SEQUENCE: 10 MetGly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15Arg Gly Ser His Met Ala Ser Pro Asp Ser Asp Asp Arg Val Thr Pro 20 25 30Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser Tyr 35 40 45Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln 50 55 60Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln 65 70 7580 Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser 85 9095 Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser Phe Asp Glu Asp 100105 110 Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr115 120 125 Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser Phe Asp GluGlu 130 135 140 Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val Met AsnArg Ala 145 150 155 160 Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu AspAsn Leu Lys Lys 165 170 175 Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg AsnGlu Asp Ala Arg Ser 180 185 190 Pro Phe Tyr Ser Ala Leu Arg Asn Thr ProSer Phe Lys Glu Arg Asn 195 200 205 Gly Gly Asn His Asp Pro Ser Arg MetLys Ala Val Ile Tyr Ser Lys 210 215 220 His Phe Trp Ser Gly Gln Asp ArgSer Ser Ser Ala Asp Lys Arg Lys 225 230 235 240 Tyr Gly Asp Pro Asp AlaPhe Arg Pro Ala Pro Gly Thr Gly Leu Val 245 250 255 Asp Met Ser Arg AspArg Asn Ile Pro Arg Ser Pro Thr Ser Pro Gly 260 265 270 Glu Gly Phe ValAsn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu 275 280 285 Ala Asp AlaAsp Lys Thr Val Trp Thr His Gly Asn His Tyr His Ala 290 295 300 Pro AsnGly Ser Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe Arg 305 310 315 320Asn Trp Ser Glu Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val Ile 325 330335 Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro Asp Lys Val Lys Gln 340345 350 Gly Trp Pro 355 <210> SEQ ID NO 11 <211> LENGTH: 1074 <212>TYPE: DNA <213> ORGANISM: Streptomyces cinnamoneus ATCC 11874 <400>SEQUENCE: 11 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcgcggcagccat 60 atggctagct cccgggcccc ctccgatgac cgggaaactc ctcccgccgagccgctcgac 120 aggatgcctg aggcgtaccg ggcctacgga ggcagggcca ctacggtcgtcaacaactac 180 atacgcaagt ggcagcaggt ctacagtcac cgcgacggaa agaaacagcaaatgaccgaa 240 gagcagcgag aaaagctgtc ctacggttgc gttggcgtca cctgggtcaactcgggcccc 300 tacccgacga acagattggc gttcgcgtcc ttcgacgaga acaagtacaagaacgacctg 360 aagaacacca gcccccgacc cgatgaaacg cgggcggagt tcgagggtcgcatcgccaag 420 ggcagtttcg acgaggggaa gggtttcaag cgggcgcgtg atgtggcgtccgtcatgaac 480 aaggccctgg aaaatgccca cgacgagggg acttacatca acaacctcaagacggagctc 540 acgaacaaca atgacgctct gctccgcgag gacagccgct cgaacttctactcggcgctg 600 aggaacacac cgtccttcaa ggaaagggac ggcggcaact acgacccgtccaagatgaag 660 gcggtgatct actcgaagca cttctggagc gggcaggacc agcggggctcctccgacaag 720 aggaagtacg gcgacccgga agccttccgc cccgaccagg gtaccggcctggtcgacatg 780 tcgaaggaca gaagcattcc gcgcagtccg gccaagcccg gcgaaggttgggtcaatttc 840 gactacggtt ggttcggggc tcaaacagaa gcggatgccg acaaaaccacatggacccac 900 ggcgaccact accacgcgcc caatagcgac ctgggcccca tgcacgtacacgagagcaag 960 ttccggaagt ggtctgccgg gtacgcggac ttcgaccgcg gagcctacgtgatcacgttc 1020 atacccaaga gctggaacac cgcccccgcc aaggtggagc aaggctggccgtga 1074 <210> SEQ ID NO 12 <211> LENGTH: 358 <212> TYPE: PRT <213>ORGANISM: Streptomyces cinnamoneus ATCC 11874 <400> SEQUENCE: 12 Met GlySer Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 ArgGly Ser His Met Ala Ser Ser Arg Ala Pro Ser Asp Asp Arg Glu 20 25 30 ThrPro Pro Ala Glu Pro Leu Asp Arg Met Pro Glu Ala Tyr Arg Ala 35 40 45 TyrGly Gly Arg Ala Thr Thr Val Val Asn Asn Tyr Ile Arg Lys Trp 50 55 60 GlnGln Val Tyr Ser His Arg Asp Gly Lys Lys Gln Gln Met Thr Glu 65 70 75 80Glu Gln Arg Glu Lys Leu Ser Tyr Gly Cys Val Gly Val Thr Trp Val 85 90 95Asn Ser Gly Pro Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser Phe Asp 100 105110 Glu Asn Lys Tyr Lys Asn Asp Leu Lys Asn Thr Ser Pro Arg Pro Asp 115120 125 Glu Thr Arg Ala Glu Phe Glu Gly Arg Ile Ala Lys Gly Ser Phe Asp130 135 140 Glu Gly Lys Gly Phe Lys Arg Ala Arg Asp Val Ala Ser Val MetAsn 145 150 155 160 Lys Ala Arg Leu Glu Asn Ala His Asp Glu Gly Thr TyrIle Asn Asn 165 170 175 Leu Lys Thr Glu Leu Thr Asn Asn Asn Asp Ala LeuLeu Arg Glu Asp 180 185 190 Ser Arg Ser Asn Phe Tyr Ser Ala Leu Arg AsnThr Pro Ser Phe Lys 195 200 205 Glu Arg Asp Gly Gly Asn Tyr Asp Pro SerLys Met Lys Ala Val Ile 210 215 220 Tyr Ser Lys His Phe Trp Ser Gly GlnAsp Gln Arg Gly Ser Ser Asp 225 230 235 240 Lys Arg Lys Tyr Gly Asp ProGlu Ala Phe Arg Pro Asp Gln Gly Thr 245 250 255 Gly Leu Val Asp Met SerLys Asp Arg Ser Ile Pro Arg Ser Pro Ala 260 265 270 Lys Pro Gly Glu GlyTrp Val Asn Phe Asp Tyr Gly Trp Phe Gly Ala 275 280 285 Gln Thr Glu AlaAsp Ala Asp Lys Thr Thr Trp Thr His Gly Asp His 290 295 300 Tyr His AlaPro Asn Ser Asp Leu Gly Pro Met His Val His Glu Ser 305 310 315 320 LysPhe Arg Lys Trp Ser Ala Gly Tyr Ala Asp Phe Asp Arg Gly Ala 325 330 335Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro Ala Lys 340 345350 Val Glu Gln Gly Trp Pro 355 <210> SEQ ID NO 13 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Humicola grisea var. thermoides ATCC16453 <220> FEATURE: <221> NAME/KEY: primer_bind <222> LOCATION:(2)..(25) <400> SEQUENCE: 13 gctagatgcg ttcctccccc ctcctc 26 <210> SEQID NO 14 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Humicolagrisea var. thermoidea ATCC 16453 <400> SEQUENCE: 14 ttacaggcactgatggtacc agtc 24 <210> SEQ ID NO 15 <211> LENGTH: 42 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 15 Asp Ala Glu Phe Arg HisAsp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe AlaGlu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val GlyGly Val Val Ile Ala 35 40 <210> SEQ ID NO 16 <211> LENGTH: 20 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthetic Bovine Serum Albumin peptide <400> SEQUENCE: 16Lys Lys Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 1 5 1015 Ser Gln Gln Gln 20

What is claimed is:
 1. A method for producing a polyvalent antigen using a biological agent, comprising: preparing an antigen in a cross-linking solution; combining the antigen cross-linking solution with a solution of the biological agent into a mixture; and incubating the mixture at a temperature for a period of time sufficient to effect cross-linking of the antigen into a plurarilty of cross-linked products; and providing the cross-linked products to be used as the polyvalent antigen.
 2. The method of claim 1, further comprising monitoring a change of color in the mixture.
 3. The method of claim 2, wherein the change of color in the mixture comprises an increase in an absorbance value (OD value) from about 0.0001 to about 0.1 or more at a wavelength from about 400 to about
 500. 4. The method of claim 2, wherein the change of color in the mixture comprises an absorbance value of about 0.2 or more at OD₄₅₀.
 5. The method of claim 1, further comprising purifying the plurarilty of cross-linked products after said incubating step.
 6. The method of claim 1, further comprising providing the polyvalent antigen to an animal.
 7. The method of claim 1, wherein the antigen is selected from a group consisting of polypeptides, naturally occurring proteins, polyamino acids, cell-membrane-associated proteins, tumor-associated antigens, cytokines, cytokine receptors, bacterial toxins, whole bacterial cells, viral coat proteins, whole viruses, viral glycoproteins, cell wall-derived coat proteins, peptides, synthetic peptides, any modification of the aforementioned compounds, and derivatives thereof.
 8. The method of claim 1, wherein the antigen in the cross-linking solution comprises two or more antigens.
 9. The method of claim 1, wherein the antigen in the cross-linking solution comprises at least one glutamine residue.
 10. The method of claim 1, wherein the antigen in the cross-linking solution comprises at least one lysine residue.
 11. The method of claim 1, wherein the biological agent is an enzyme selected from a group consisting of transglutaminases, oxidases, sulfhydryl oxidases, lipoxygenases, polyphenol oxidases, tyrosinase, laccases, lysyl oxidases, peroxidase, isomerases, protein disulfide-isomerases, reductases, protein-disulfide reductases, and combinations thereof.
 12. The method of claim 1, wherein the biological agent is a transglutaminase.
 13. The method of claim 1, wherein the biological agent is a recombinant microbial transglutaminase.
 14. The method of claim 1, wherein the biological agent is purified from an organism selected from the group consisting of Streptomyces mobaraensis, Streptomyces cinnamoneus, and isolates thereof.
 15. The method of claim 1, wherein the cross-linking solution comprises at least one reducing agent, deionized water, a pH-buffering agent for adjusting the pH of the cross-linking solution, and combinations thereof.
 16. The method of claim 15, wherein the cross-linking solution further comprises up to about 50% of glycerol.
 17. The method of claim 15, wherein the cross-linking solution further comprises phosphate-buffered saline solution (PBS).
 18. The method of claim 15, wherein the pH of the composition is from about 5 to about
 11. 19. The method of claim 15, wherein the at least one reducing agent comprises up to about 0.5 M DTT.
 20. The method of claim 15, wherein the cross-linking solution comprises about 10 mM DTT, about 20% to about 30% of glycerol, about 50 mM Tris base titrated to a pH of about 6 to about
 9. 21. A polyvalent antigen prepared in accordance with the method of claim
 1. 22. The polyvalent antigen of claim 21, wherein the antigen is selected from a group consisting of β-casein, serum albumin, cellulase, bovine serum albumin, Histone H3, glucose oxidase, ovalbumin, β-amyloid peptide, synthetic peptides, peptides having glutamine and lysine residues, and combinations of.
 23. The polyvalent antigen of claim 21, wherein the antigen is a mixture of two or more antigens.
 24. A purified antibody that binds specifically to the polyvalent antigen of claim
 1. 25. The purified antibody of claim 24, wherein the titer of the purified antibody against the antigen is about 1000 or more by an ELISA assay.
 26. A pharmaceutical composition comprising the polyvalent antigen of claim
 1. 27. A method for producing a polyvalent antigen using a transglutaminase, comprising: preparing an antigen in a cross-linking solution; combining the antigen cross-linking solution with a solution of the transglutaminase into a mixture; incubating the mixture for a period of time at a temperature sufficient to effect cross-linking of the antigen into a plurality of cross-linked products; and providing the cross-linked products to be used as the polyvalent antigen.
 28. The method of claim 27, further comprising monitoring a change of color in the mixture.
 29. The method of claim 27, further comprising purifying the plurality of cross-linked products after said incubating step.
 30. The method of claim 27, further comprising providing the plurality of cross-linked products to an animal.
 31. The method of claim 27, wherein the antigen is selected from a group consisting of polypeptides, naturally occurring proteins, polyamino acids, cell-membrane-associated proteins, tumor-associated antigens, cytokines, cytokine receptors, bacterial toxins, whole bacterial cells, viral coat proteins, whole viruses, viral glycoproteins, cell wall-derived coat proteins, peptides, synthetic peptides, any modification of the aforementioned compounds, and derivatives thereof.
 32. The method of claim 27, wherein the antigen in the cross-linking solution comprises two or more antigens.
 33. The method of claim 27, wherein the antigen in the cross-linking solution comprises at least one glutamine residue.
 34. The method of claim 27, wherein the transglutaminase comprises recombinant microbial transglutaminase.
 35. A polyvalent antigen composition prepared in accordance with the method of claim
 27. 36. A purified antibody that binds specifically to the polyvalent antigen of claim
 27. 37. A pharmaceutical composition comprising the polyvalent antigen of claim
 27. 38. A method for preparing an antigen having immunogenic activity, comprising: preparing a compound in a cross-linking solution; combining the compound cross-linking solution with a biological agent into a mixture; incubating the mixture at a temperature for a period of time sufficient to effect cross-linking of the compound into a plurality of cross-linked products; and providing the cross-linked products to be used as the antigen.
 39. The method of claim 38, wherein the compound is selected from a group consisting of polypeptides, naturally occurring proteins, polyamino acids, cell-membrane-associated proteins, tumor-associated antigens, cytokines, cytokine receptors, bacterial toxins, whole bacterial cells, viral coat proteins, whole viruses, viral glycoproteins, cell wall-derived coat proteins, peptides, synthetic peptides, any modification of the aforementioned compounds, and derivatives thereof.
 40. The method of claim 38, wherein the biological agent is a recombinant microbial transglutaminase.
 41. A method for preparing an antigen having immunogenic activity, comprising: preparing two or more compounds; combining the two or more compounds in a cross-linking solution with a biological agent into a mixture; incubating the mixture at a temperature for a period of time sufficient to effect cross-linking of the two or more compounds into a plurality of cross-linked products; and providing the cross-linked products to be used as the antigen.
 42. The method of claim 41, wherein the compound is selected from a group consisting of polypeptides, naturally occurring proteins, polyamino acids, cell-membrane-associated proteins, tumor-associated antigens, cytokines, cytokine receptors, bacterial toxins, whole bacterial cells, viral coat proteins, whole viruses, viral glycoproteins, cell wall-derived coat proteins, peptides, synthetic peptides, any modification of the aforementioned compounds, and derivatives thereof.
 43. The method of claim 41, wherein the biological agent is a recombinant microbial transglutaminase.
 44. A method for preparing a vaccine having immunogenic activity, comprising: preparing a compound in a cross-linking solution; combining the compound cross-linking solution with a biological agent into a mixture; incubating the mixture for a period of time at a temperature sufficient to effect cross-linking of the compound into a plurality of cross-linked products; and providing the plurality of cross-linked products to be used as the vaccine.
 45. The method of claim 44, further comprising immunizing an animal with the vaccine.
 46. The method of claim 45, further comprising isolating antibodies produced by the animal.
 47. The method of claim 46, further comprising screening the isolated antibodies with the compound, thereby identifying an antibody that binds specifically to the compound.
 48. The method of claim 44, further comprising providing another compound selected from the group consisting of conjugate compound, co-stimulating factor for immune response, DNA vaccine, adjuvants, and combinations thereof, together with the cross-linked products to be used as the vaccine, for promoting immune responses.
 49. The method of claim 44, further comprising providing another compound selected from the group consisting of conjugate compound, co-stimulating factor for immune response, DNA vaccine, adjuvants, and combinations thereof, after the cross-linked products used as the vaccine, for promoting immune responses.
 50. The method of claim 44, wherein the compound is selected from a group consisting of polypeptides, naturally occurring proteins, polyamino acids, cell-membrane-associated proteins, tumor-associated antigens, cytokines, cytokine receptors, bacterial toxins, whole bacterial cells, viral coat proteins, whole viruses, viral glycoproteins, cell wall-derived coat proteins, peptides, synthetic peptides, any modification of the aforementioned compounds, and derivatives thereof.
 51. The method of claim 44, wherein the compound in the cross-linking solution comprises two or more compounds.
 52. The method of claim 44, wherein the compound in the cross-linking solution comprises at least one glutamine residue.
 53. The method of claim 44, wherein the compound in the cross-linking solution comprises one or more tumor-associated antigens.
 54. The method of claim 44, wherein the compound in the cross-linking solution comprises one or more Alzheimer's related polypeptides.
 55. The method of claim 44, wherein the compound in the cross-linking solution comprises one or more viral glycoproteins.
 56. The method of claim 44, wherein the compound comprises one or more attenuated infectious agents.
 57. The method of claim 44, wherein the biological agent is a recombinant microbial transglutaminase.
 58. A method for producing an antibody specifically for a compound, comprising: preparing the compound in a cross-linking solution; combining the compound cross-linking solution with a biological agent into a mixture; incubating the mixture at a temperature for a period of time sufficient to effect cross-linking of the compound into a plurality of cross-linked products; immunizing an animal with the plurality of cross-linked products under conditions to elicit an antibody response; and isolating antibodies produced by the animal.
 59. The method of claim 58, further comprising purifying the plurality of cross-linked products after said incubating step.
 60. The method of claim 58, further comprising screening the isolated antibodies with the compound, thereby identifying an antibody that binds specifically to the compound.
 61. The method of claim 58, wherein the isolated antibodies comprise a titer of about 1000 or more against the compound by an ELISA assay.
 62. The method of claim 58, wherein the compound is selected from a group consisting of polypeptides, naturally occurring proteins, polyamino acids, cell-membrane-associated proteins, tumor-associated antigens, cytokines, cytokine receptors, bacterial toxins, whole bacterial cells, viral coat proteins, whole viruses, viral glycoproteins, cell wall-derived coat proteins, peptides, synthetic peptides, any modification of the aforementioned compounds, and derivatives thereof.
 63. The method of claim 58, wherein the compound in the cross-linking solution comprises two or more compounds.
 64. The method of claim 58, wherein the compound in the cross-linking solution comprises at least one glutamine residue.
 65. The method of claim 58, wherein the biological agent is a recombinant microbial transglutaminase.
 66. An antigen composition, comprising: a plurality of cross-linked products prepared by combining a compound with a solution of a biological agent in a cross-linking solution into a mixture at a temperature for a period of sufficient time.
 67. The antigen composition of claim 66, further comprising another compound selected from the group consisting of conjugate compound, co-stimulating factor for immune response, DNA vaccine, adjuvants, and combinations thereof, for promoting immune responses.
 68. The antigen composition of claim 66, wherein the compound is selected from a group consisting of polypeptides, naturally occurring proteins, polyamino acids, cell-membrane-associated proteins, tumor-associated antigens, cytokines, cytokine receptors, bacterial toxins, whole bacterial cells, viral coat proteins, whole viruses, viral glycoproteins, cell wall-derived coat proteins, peptides, synthetic peptides, any modification of the aforementioned compounds, and derivatives thereof.
 69. The antigen composition of claim 66, wherein the compound in the cross-linking solution comprises at least one glutamine residue.
 70. The antigen composition of claim 66, wherein the compound in the cross-linking solution comprises two or more compounds.
 71. The antigen composition of claim 66, wherein the biological agent is selected from a group consisting of transglutaminases, oxidases, sulfhydryl oxidases, lipoxygenases, polyphenol oxidases, tyrosinase, laccases, lysyl oxidases, peroxidase, isomerases, protein disulfide-isomerases, reductases, protein-disulfide reductases, and combinations thereof.
 72. The antigen composition of claim 66, wherein the biological agent is a recombinant microbial transglutaminase.
 73. The antigen composition of claim 66, further comprising monitoring a change of color in the mixture, wherein the change of color in the mixture comprises an increase in an absorbance value (OD value) from about 0.0001 to about 0.1 or more at a wavelength from about 400 to about
 500. 74. A cross-linking solution composition for cross-linking a polypeptide by an biological agent, comprising: at least one reducing agent; up to about 50% of glycerol; deionized water; and a pH buffering agent for adjusting the pH of the composition.
 75. The cross-linking solution composition of claim 74, further comprises phosphate-buffered saline solution (PBS).
 76. The cross-linking solution composition of claim 74, wherein the pH of the composition is from about 5 to about
 11. 77. The cross-linking solution composition of claim 74, wherein the at least one reducing agent comprises up to about 0.5 M DTT.
 78. The cross-linking solution composition of claim 74, wherein the composition comprises about 10 mM DTT, about 20% to about 30% of glycerol, about 50 mM Tris buffer, and a pH of about 6 to about
 9. 79. The cross-linking solution composition of claim 74, wherein the biological agent is a transglutaminase.
 80. The cross-linking solution composition of claim 74, wherein the biological agent is a recombinant microbial transglutaminase. 